An adaptive stress response that confers cellular resilience to decreased ubiquitination.

Ubiquitination is a post-translational modification initiated by the E1 enzyme UBA1, which transfers ubiquitin to ~35 E2 ubiquitin-conjugating enzymes. While UBA1 loss is cell lethal, it remains unknown how partial reduction in UBA1 activity is endured. Here, we utilize deep-coverage mass spectrometry to define the E1-E2 interactome and to determine the proteins that are modulated by knockdown of UBA1 and of each E2 in human cells. These analyses define the UBA1/E2-sensitive proteome and the E2 specificity in protein modulation. Interestingly, profound adaptations in peroxisomes and other organelles are triggered by decreased ubiquitination. While the cargo receptor PEX5 depends on its mono-ubiquitination for binding to peroxisomal proteins and importing them into peroxisomes, we find that UBA1/E2 knockdown induces the compensatory upregulation of other PEX proteins necessary for PEX5 docking to the peroxisomal membrane. Altogether, this study defines a homeostatic mechanism that sustains peroxisomal protein import in cells with decreased ubiquitination capacity.

Mutual antagonism between glucocorticoid and canonical Wnt signaling pathways in B-cell acute lymphoblastic leukemia.

Glucocorticoids (GCs; i.e., steroids) are important chemotherapeutic agents in the treatment of B-cell precursor acute lymphoblastic leukemia (B-ALL) and de novo GC resistance predicts relapse and poor clinical outcome in patients. Glucocorticoids induce B-ALL cell apoptosis through activation of glucocorticoid receptor (GR), a ligand-induced nuclear receptor transcription factor (TF). We previously identified disruptions to glucocorticoid receptor (GR)-bound cis -regulatory elements controlling TLE1 expression in GC-resistant primary B-ALL cells from patients. TLE1 is a GC-response gene up-regulated by steroids and functions as a canonical Wnt signaling repressor. To better understand the mechanistic relationship between GC signaling and canonical Wnt signaling, we performed diverse functional analyses that identified extensive crosstalk and mutual antagonism between these two signaling pathways in B-ALL. We determined that crosstalk and antagonism was driven by the binding of GR and the canonical Wnt signaling TFs LEF1 and TCF7L2 to overlapping sets of cis -regulatory elements associated with genes impacting cell death and cell proliferation, and was further accompanied by overlapping and opposing transcriptional programs. Our data additionally suggest that cis -regulatory disruptions at TLE1 are linked to GC resistance through a dampening of the GC response and GC-mediated apoptosis via enhanced canonical Wnt signaling. As a result of the extensive genomic and gene regulatory connectivity between these two signaling pathways, our data supports the importance of canonical Wnt signaling in mediating GC resistance in B-ALL.

Identification of Potent, Selective, and Orally Bioavailable Small-Molecule GSPT1/2 Degraders from a Focused Library of Cereblon Modulators.

Whereas the PROTAC approach to target protein degradation greatly benefits from rational design, the discovery of small-molecule degraders relies mostly on phenotypic screening and retrospective target identification efforts. Here, we describe the design, synthesis, and screening of a large diverse library of thalidomide analogues against a panel of patient-derived leukemia and medulloblastoma cell lines. These efforts led to the discovery of potent and novel GSPT1/2 degraders displaying selectivity over classical IMiD neosubstrates, such as IKZF1/3, and high oral bioavailability in mice. Taken together, this study offers compound 6 (SJ6986) as a valuable chemical probe for studying the role of GSPT1/2 in vitro and in vivo, and it supports the utility of a diverse library of CRBN binders in the pursuit of targeting undruggable oncoproteins.

High-throughput and Deep-proteome Profiling by 16-plex Tandem Mass Tag Labeling Coupled with Two-dimensional Chromatography and Mass Spectrometry.

Isobaric tandem mass tag (TMT) labeling is widely used in proteomics because of its high multiplexing capacity and deep proteome coverage. Recently, an expanded 16-plex TMT method has been introduced, which further increases the throughput of proteomic studies. In this manuscript, we present an optimized protocol for 16-plex TMT-based deep-proteome profiling, including protein sample preparation, enzymatic digestion, TMT labeling reaction, two-dimensional reverse-phase liquid chromatography (LC/LC) fractionation, tandem mass spectrometry (MS/MS), and computational data processing. The crucial quality control steps and improvements in the process specific for the 16-plex TMT analysis are highlighted. This multiplexed process offers a powerful tool for profiling a variety of complex samples such as cells, tissues, and clinical specimens. More than 10,000 proteins and posttranslational modifications such as phosphorylation, methylation, acetylation, and ubiquitination in highly complex biological samples from up to 16 different samples can be quantified in a single experiment, providing a potent tool for basic and clinical research.

Toxicoproteomic Profiling of hPXR Transgenic Mice Treated with Rifampicin and Isoniazid.

Tuberculosis is a global health threat that affects millions of people every year, and treatment-limiting toxicity remains a considerable source of treatment failure. Recent reports have characterized the nature of hPXR-mediated hepatotoxicity and the systemic toxicity of antitubercular drugs. The antitubercular drug isoniazid plays a role in such pathologic states as acute intermittent porphyria, anemia, hepatotoxicity, hypercoagulable states (deep vein thrombosis, pulmonary embolism, or ischemic stroke), pellagra (vitamin B3 deficiency), peripheral neuropathy, and vitamin B6 deficiency. However, the mechanisms by which isoniazid administration leads to these states are unclear. To elucidate the mechanism of rifampicin- and isoniazid-induced liver and systemic injury, we performed tandem mass tag mass spectrometry-based proteomic screening of mPxr-/- and hPXR mice treated with combinations of rifampicin and isoniazid. Proteomic profiling analysis suggested that the hPXR liver proteome is affected by antitubercular therapy to disrupt [Fe-S] cluster assembly machinery, [2Fe-2S] cluster-containing proteins, cytochrome P450 enzymes, heme biosynthesis, homocysteine catabolism, oxidative stress responses, vitamin B3 metabolism, and vitamin B6 metabolism. These novel findings provide insight into the etiology of some of these processes and potential targets for subsequent investigations. Data are available via ProteomeXchange with identifier PXD019505.

A Cancer-Specific Ubiquitin Ligase Drives mRNA Alternative Polyadenylation by Ubiquitinating the mRNA 3' End Processing Complex.

Alternative polyadenylation (APA) contributes to transcriptome complexity by generating mRNA isoforms with varying 3' UTR lengths. APA leading to 3' UTR shortening (3' US) is a common feature of most cancer cells; however, the molecular mechanisms are not understood. Here, we describe a widespread mechanism promoting 3' US in cancer through ubiquitination of the mRNA 3' end processing complex protein, PCF11, by the cancer-specific MAGE-A11-HUWE1 ubiquitin ligase. MAGE-A11 is normally expressed only in the male germline but is frequently re-activated in cancers. MAGE-A11 is necessary for cancer cell viability and is sufficient to drive tumorigenesis. Screening for targets of MAGE-A11 revealed that it ubiquitinates PCF11, resulting in loss of CFIm25 from the mRNA 3' end processing complex. This leads to APA of many transcripts affecting core oncogenic and tumor suppressors, including cyclin D2 and PTEN. These findings provide insights into the molecular mechanisms driving APA in cancer and suggest therapeutic strategies.

Regulation of MAGE-A3/6 by the CRL4-DCAF12 ubiquitin ligase and nutrient availability.

Melanoma antigen genes (MAGEs) are emerging as important oncogenic drivers that are normally restricted to expression in male germ cells but are aberrantly expressed in cancers and promote tumorigenesis. Mechanistically, MAGEs function as substrate specifying subunits of E3 ubiquitin ligases. Thus, the activation of germline-specific genes in cancer can drive metabolic and signaling pathways through altered ubiquitination to promote tumorigenesis. However, the mechanisms regulating MAGE expression and activity are unclear. Here, we describe how the MAGE-A3/6 proteins that function as repressors of autophagy are downregulated in response to nutrient deprivation. Short-term cellular starvation promotes rapid MAGE-A3/6 degradation in a proteasome-dependent manner. Proteomic analysis reveals that degradation of MAGE-A3/6 is controlled by the CRL4-DCAF12 E3 ubiquitin ligase. Importantly, the degradation of MAGE-A3/6 by CRL4-DCAF12 is required for starvation-induced autophagy. These findings suggest that oncogenic MAGEs can be dynamically controlled in response to stress to allow cellular adaptation, autophagy regulation, and tumor growth and that CRL4-DCAF12 activity is responsive to nutrient status.

Mutant LRRK2 mediates peripheral and central immune responses leading to neurodegeneration in vivo.

Missense mutations in the leucine rich repeat kinase 2 (LRRK2) gene result in late-onset Parkinson's disease. The incomplete penetrance of LRRK2 mutations in humans and LRRK2 murine models of Parkinson's disease suggests that the disease may result from a complex interplay of genetic predispositions and persistent exogenous insults. Since neuroinflammation is commonly associated with the pathogenesis of Parkinson's disease, we examine a potential role of mutant LRRK2 in regulation of the immune response and inflammatory signalling in vivo. Here, we show that mice overexpressing human pathogenic LRRK2 mutations, but not wild-type mice or mice overexpressing human wild-type LRRK2 exhibit long-term lipopolysaccharide-induced nigral neuronal loss. This neurodegeneration is accompanied by an exacerbated neuroinflammation in the brain. The increased immune response in the brain of mutant mice subsequently has an effect on neurons by inducing intraneuronal LRRK2 upregulation. However, the enhanced neuroinflammation is unlikely to be triggered by dysfunctional microglia or infiltrated T cells and/or monocytes, but by peripheral circulating inflammatory molecules. Analysis of cytokine kinetics and inflammatory pathways in the peripheral immune cells demonstrates that LRRK2 mutation alters type II interferon immune response, suggesting that this increased neuroinflammatory response may arise outside the central nervous system. Overall, this study suggests that peripheral immune signalling plays an unexpected-but important-role in the regulation of neurodegeneration in LRRK2-associated Parkinson's disease, and provides new targets for interfering with the onset and progression of the disease.

Extensive Peptide Fractionation and y1 Ion-Based Interference Detection Method for Enabling Accurate Quantification by Isobaric Labeling and Mass Spectrometry.

Isobaric labeling quantification by mass spectrometry (MS) has emerged as a powerful technology for multiplexed large-scale protein profiling, but measurement accuracy in complex mixtures is confounded by the interference from coisolated ions, resulting in ratio compression. Here we report that the ratio compression can be essentially resolved by the combination of pre-MS peptide fractionation, MS2-based interference detection, and post-MS computational interference correction. To recapitulate the complexity of biological samples, we pooled tandem mass tag (TMT)-labeled Escherichia coli peptides at 1:3:10 ratios and added in ∼20-fold more rat peptides as background, followed by the analysis of two-dimensional liquid chromatography (LC)-MS/MS. Systematic investigation shows that quantitative interference was impacted by LC fractionation depth, MS isolation window, and peptide loading amount. Exhaustive fractionation (320 × 4 h) can nearly eliminate the interference and achieve results comparable to the MS3-based method. Importantly, the interference in MS2 scans can be estimated by the intensity of contaminated y1 product ions, and we thus developed an algorithm to correct reporter ion ratios of tryptic peptides. Our data indicate that intermediate fractionation (40 × 2 h) and y1 ion-based correction allow accurate and deep TMT profiling of more than 10 000 proteins, which represents a straightforward and affordable strategy in isobaric labeling proteomics.

Efficacy of Retinoids in IKZF1-Mutated BCR-ABL1 Acute Lymphoblastic Leukemia.

Alterations of IKZF1, encoding the lymphoid transcription factor IKAROS, are a hallmark of high-risk acute lymphoblastic leukemia (ALL), however the role of IKZF1 alterations in ALL pathogenesis is poorly understood. Here, we show that in mouse models of BCR-ABL1 leukemia, Ikzf1 and Arf alterations synergistically promote the development of an aggressive lymphoid leukemia. Ikzf1 alterations result in acquisition of stem cell-like features, including self-renewal and increased bone marrow stromal adhesion. Retinoid receptor agonists reversed this phenotype, partly by inducing expression of IKZF1, resulting in abrogation of adhesion and self-renewal, cell cycle arrest, and attenuation of proliferation without direct cytotoxicity. Retinoids potentiated the activity of dasatinib in mouse and human BCR-ABL1 ALL, providing an additional therapeutic option in IKZF1-mutated ALL.

Quantitative protein analysis by mass spectrometry.

Mass spectrometry is one of the most sensitive methods in analytical chemistry, and its application in proteomics has been rapidly expanded after sequencing the human genome. Mass spectrometry is now the mainstream approach for identification and quantification of proteins and posttranslational modifications, either in small scale or in the entire proteome. Shotgun proteomics can analyze up to 10,000 proteins in a comprehensive study, with detection sensitivity in the picogram range. In this chapter, we describe major experimental steps in a shotgun proteomics platform, including sample preparation in the context of studying protein-protein interaction, mass spectrometric data acquisition, and database search to identify proteins and posttranslational modification analysis. Proteome quantification strategies and bioinformatics analysis are also illustrated. Finally, we discuss the capabilities, limitations, and potential improvements of current platforms.

HECT-E3 ligase ETC-1 regulates securin and cyclin B1 cytoplasmic abundance to promote timely anaphase during meiosis in C. elegans.

The anaphase inhibitor securin plays a crucial role in regulating the timing of sister chromatid separation during mitosis. When sister chromatid pairs become bioriented, the E3 ligase anaphase promoting complex/cyclosome (APC/C) ubiquitylates securin for proteolysis, triggering sister chromatid separation. Securin is also implicated in regulating meiotic progression. Securin protein levels change sharply during cell cycle progression, enabling its timely action. To understand the mechanism underlying the tightly regulated dynamics of securin, we analyzed the subcellular localization of the securin IFY-1 during C. elegans development. IFY-1 was highly expressed in the cytoplasm of germ cells. The cytoplasmic level of IFY-1 declined immediately following meiosis I division and remained low during meiosis II and following mitoses. We identified a C. elegans homolog of another type of E3 ligase, UBE3C, designated ETC-1, as a regulator of the cytoplasmic IFY-1 level. RNAi-mediated depletion of ETC-1 stabilized IFY-1 and CYB-1 (cyclin B1) in post-meiosis I embryos. ETC-1 knockdown in a reduced APC function background caused an embryonic lethal phenotype. In vitro, ETC-1 ubiquitylates IFY-1 and CYB-1 in the presence of the E2 enzyme UBC-18, which functions in pharyngeal development. Genetic analysis revealed that UBC-18 plays a distinct role together with ETC-1 in regulating the cytoplasmic level of IFY-1 during meiosis. Our study reports a novel mechanism, mediated by ETC-1, that co-operates with APC/C to maintain the meiotic arrest required for proper cell cycle timing during reproduction.