Publications by authors named "Vishwajeeth Pagala"

26 Publications

  • Page 1 of 1

An age-downregulated ribosomal RpS28 protein variant regulates the muscle proteome.

G3 (Bethesda) 2021 May 11. Epub 2021 May 11.

Department of Developmental Neurobiology, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA.

Recent evidence indicates that the composition of the ribosome is heterogeneous and that multiple types of specialized ribosomes regulate the synthesis of specific protein subsets. In Drosophila, we find that expression of the ribosomal RpS28 protein variants RpS28a and RpS28-like preferentially occurs in the germline, a tissue resistant to aging, and that it significantly declines in skeletal muscle during aging. Muscle-specific overexpression of RpS28a at levels similar to those seen in the germline decreases early mortality and promotes the synthesis of a subset of proteins with known anti-aging roles, some of which have preferential expression in the germline. These findings indicate a contribution of specialized ribosomal proteins to the regulation of the muscle proteome during aging.
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http://dx.doi.org/10.1093/g3journal/jkab165DOI Listing
May 2021

Integrative network analysis reveals USP7 haploinsufficiency inhibits E-protein activity in pediatric T-lineage acute lymphoblastic leukemia (T-ALL).

Sci Rep 2021 Mar 4;11(1):5154. Epub 2021 Mar 4.

Department of Computational Biology, St Jude Children's Research Hospital, 262 Danny Thomas Place, MS321, Memphis, TN, 38105, USA.

USP7, which encodes a deubiquitylating enzyme, is among the most frequently mutated genes in pediatric T-ALL, with somatic heterozygous loss-of-function mutations (haploinsufficiency) predominantly affecting the subgroup that has aberrant TAL1 oncogene activation. Network analysis of > 200 T-ALL transcriptomes linked USP7 haploinsufficiency with decreased activities of E-proteins. E-proteins are also negatively regulated by TAL1, leading to concerted down-regulation of E-protein target genes involved in T-cell development. In T-ALL cell lines, we showed the physical interaction of USP7 with E-proteins and TAL1 by mass spectrometry and ChIP-seq. Haploinsufficient but not complete CRISPR knock-out of USP7 showed accelerated cell growth and validated transcriptional down-regulation of E-protein targets. Our study unveiled the synergistic effect of USP7 haploinsufficiency with aberrant TAL1 activation on T-ALL, implicating USP7 as a haploinsufficient tumor suppressor in T-ALL. Our findings caution against a universal oncogene designation for USP7 while emphasizing the dosage-dependent consequences of USP7 inhibitors currently under development as potential cancer therapeutics.
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http://dx.doi.org/10.1038/s41598-021-84647-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7933146PMC
March 2021

Antagonistic control of myofiber size and muscle protein quality control by the ubiquitin ligase UBR4 during aging.

Nat Commun 2021 03 3;12(1):1418. Epub 2021 Mar 3.

Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, TN, USA.

Sarcopenia is a degenerative condition that consists in age-induced atrophy and functional decline of skeletal muscle cells (myofibers). A common hypothesis is that inducing myofiber hypertrophy should also reinstate myofiber contractile function but such model has not been extensively tested. Here, we find that the levels of the ubiquitin ligase UBR4 increase in skeletal muscle with aging, and that UBR4 increases the proteolytic activity of the proteasome. Importantly, muscle-specific UBR4 loss rescues age-associated myofiber atrophy in mice. However, UBR4 loss reduces the muscle specific force and accelerates the decline in muscle protein quality that occurs with aging in mice. Similarly, hypertrophic signaling induced via muscle-specific loss of UBR4/poe and of ESCRT members (HGS/Hrs, STAM, USP8) that degrade ubiquitinated membrane proteins compromises muscle function and shortens lifespan in Drosophila by reducing protein quality control. Altogether, these findings indicate that these ubiquitin ligases antithetically regulate myofiber size and muscle protein quality control.
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http://dx.doi.org/10.1038/s41467-021-21738-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7930053PMC
March 2021

Deep Profiling of Microgram-Scale Proteome by Tandem Mass Tag Mass Spectrometry.

J Proteome Res 2021 01 11;20(1):337-345. Epub 2020 Nov 11.

Departments of Structural Biology and Developmental Neurobiology, Saint Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, Tennessee 38105, United States.

Tandem mass tag (TMT)-based mass spectrometry (MS) enables deep proteomic profiling of more than 10,000 proteins in complex biological samples but requires up to 100 μg protein in starting materials during a standard analysis. Here, we present a streamlined protocol to quantify more than 9000 proteins with 0.5 μg protein per sample by 16-plex TMT coupled with two-dimensional liquid chromatography and tandem mass spectrometry (LC/LC-MS/MS). In this protocol, we optimized multiple conditions to reduce sample loss, including processing each sample in a single tube to minimize surface adsorption, increasing digestion enzymes to shorten proteolysis and function as carriers, eliminating a desalting step between digestion and TMT labeling, and developing miniaturized basic pH LC for prefractionation. By profiling 16 identical human brain tissue samples of Alzheimer's disease (AD), vascular dementia (VaD), and non-dementia controls, we directly compared this new microgram-scale protocol to the standard-scale protocol, quantifying 9116 and 10,869 proteins, respectively. Importantly, bioinformatics analysis indicated that the microgram-scale protocol had adequate sensitivity and reproducibility to detect differentially expressed proteins in disease-related pathways. Thus, this newly developed protocol is of general application for deep proteomics analysis of biological and clinical samples at sub-microgram levels.
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http://dx.doi.org/10.1021/acs.jproteome.0c00426DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8262114PMC
January 2021

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

J Vis Exp 2020 08 18(162). Epub 2020 Aug 18.

Departments of Structural Biology and Developmental Neurobiology, St. Jude Children's Research Hospital; Center for Proteomics and Metabolomics, St. Jude Children's Research Hospital;

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.
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http://dx.doi.org/10.3791/61684DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7752892PMC
August 2020

Ybx1 fine-tunes PRC2 activities to control embryonic brain development.

Nat Commun 2020 08 13;11(1):4060. Epub 2020 Aug 13.

Department of Developmental Neurobiology, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN, 38105, USA.

Chromatin modifiers affect spatiotemporal gene expression programs that underlie organismal development. The Polycomb repressive complex 2 (PRC2) is a crucial chromatin modifier in executing neurodevelopmental programs. Here, we find that PRC2 interacts with the nucleic acid-binding protein Ybx1. In the mouse embryo in vivo, Ybx1 is required for forebrain specification and restricting mid-hindbrain growth. In neural progenitor cells (NPCs), Ybx1 controls self-renewal and neuronal differentiation. Mechanistically, Ybx1 highly overlaps PRC2 binding genome-wide, controls PRC2 distribution, and inhibits H3K27me3 levels. These functions are consistent with Ybx1-mediated promotion of genes involved in forebrain specification, cell proliferation, or neuronal differentiation. In Ybx1-knockout NPCs, H3K27me3 reduction by PRC2 enzymatic inhibitor or genetic depletion partially rescues gene expression and NPC functions. Our findings suggest that Ybx1 fine-tunes PRC2 activities to regulate spatiotemporal gene expression in embryonic neural development and uncover a crucial epigenetic mechanism balancing forebrain-hindbrain lineages and self-renewal-differentiation choices in NPCs.
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http://dx.doi.org/10.1038/s41467-020-17878-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7426271PMC
August 2020

Deep Multilayer Brain Proteomics Identifies Molecular Networks in Alzheimer's Disease Progression.

Neuron 2020 03 8;105(6):975-991.e7. Epub 2020 Jan 8.

Departments of Psychiatry and Neuroscience, The Alzheimer's Disease Research Center, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Mental Illness Research, Education and Clinical Center (MIRECC), James J. Peters VA Medical Center, Bronx, NY 10468, USA.

Alzheimer's disease (AD) displays a long asymptomatic stage before dementia. We characterize AD stage-associated molecular networks by profiling 14,513 proteins and 34,173 phosphosites in the human brain with mass spectrometry, highlighting 173 protein changes in 17 pathways. The altered proteins are validated in two independent cohorts, showing partial RNA dependency. Comparisons of brain tissue and cerebrospinal fluid proteomes reveal biomarker candidates. Combining with 5xFAD mouse analysis, we determine 15 Aβ-correlated proteins (e.g., MDK, NTN1, SMOC1, SLIT2, and HTRA1). 5xFAD shows a proteomic signature similar to symptomatic AD but exhibits activation of autophagy and interferon response and lacks human-specific deleterious events, such as downregulation of neurotrophic factors and synaptic proteins. Multi-omics integration prioritizes AD-related molecules and pathways, including amyloid cascade, inflammation, complement, WNT signaling, TGF-β and BMP signaling, lipid metabolism, iron homeostasis, and membrane transport. Some Aβ-correlated proteins are colocalized with amyloid plaques. Thus, the multilayer omics approach identifies protein networks during AD progression.
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http://dx.doi.org/10.1016/j.neuron.2019.12.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7318843PMC
March 2020

Deep multiomics profiling of brain tumors identifies signaling networks downstream of cancer driver genes.

Nat Commun 2019 08 16;10(1):3718. Epub 2019 Aug 16.

Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, TN, 38105, USA.

High throughput omics approaches provide an unprecedented opportunity for dissecting molecular mechanisms in cancer biology. Here we present deep profiling of whole proteome, phosphoproteome and transcriptome in two high-grade glioma (HGG) mouse models driven by mutated RTK oncogenes, PDGFRA and NTRK1, analyzing 13,860 proteins and 30,431 phosphosites by mass spectrometry. Systems biology approaches identify numerous master regulators, including 41 kinases and 23 transcription factors. Pathway activity computation and mouse survival indicate the NTRK1 mutation induces a higher activation of AKT downstream targets including MYC and JUN, drives a positive feedback loop to up-regulate multiple other RTKs, and confers higher oncogenic potency than the PDGFRA mutation. A mini-gRNA library CRISPR-Cas9 validation screening shows 56% of tested master regulators are important for the viability of NTRK-driven HGG cells, including TFs (Myc and Jun) and metabolic kinases (AMPKa1 and AMPKa2), confirming the validity of the multiomics integrative approaches, and providing novel tumor vulnerabilities.
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http://dx.doi.org/10.1038/s41467-019-11661-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6697699PMC
August 2019

A Key Role for the Ubiquitin Ligase UBR4 in Myofiber Hypertrophy in Drosophila and Mice.

Cell Rep 2019 07;28(5):1268-1281.e6

Division of Developmental Biology, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA; Department of Developmental Neurobiology, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA. Electronic address:

Skeletal muscle cell (myofiber) atrophy is a detrimental component of aging and cancer that primarily results from muscle protein degradation via the proteasome and ubiquitin ligases. Transcriptional upregulation of some ubiquitin ligases contributes to myofiber atrophy, but little is known about the role that most other ubiquitin ligases play in this process. To address this question, we have used RNAi screening in Drosophila to identify the function of > 320 evolutionarily conserved ubiquitin ligases in myofiber size regulation in vivo. We find that whereas RNAi for some ubiquitin ligases induces myofiber atrophy, loss of others (including the N-end rule ubiquitin ligase UBR4) promotes hypertrophy. In Drosophila and mouse myofibers, loss of UBR4 induces hypertrophy via decreased ubiquitination and degradation of a core set of target proteins, including the HAT1/RBBP4/RBBP7 histone-binding complex. Together, this study defines the repertoire of ubiquitin ligases that regulate myofiber size and the role of UBR4 in myofiber hypertrophy.
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http://dx.doi.org/10.1016/j.celrep.2019.06.094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6697171PMC
July 2019

Circadian gene variants and the skeletal muscle circadian clock contribute to the evolutionary divergence in longevity across populations.

Genome Res 2019 08 27;29(8):1262-1276. Epub 2019 Jun 27.

Division of Developmental Biology, Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.

Organisms use endogenous clocks to adapt to the rhythmicity of the environment and to synchronize social activities. Although the circadian cycle is implicated in aging, it is unknown whether natural variation in its function contributes to differences in lifespan between populations and whether the circadian clock of specific tissues is key for longevity. We have sequenced the genomes of strains with exceptional longevity that were obtained via multiple rounds of selection from a parental strain. Comparison of genomic, transcriptomic, and proteomic data revealed that changes in gene expression due to intergenic polymorphisms are associated with longevity and preservation of skeletal muscle function with aging in these strains. Analysis of transcription factors differentially modulated in long-lived versus parental strains indicates a possible role of circadian clock core components. Specifically, there is higher and and lower expression in the muscle of strains with delayed aging compared to the parental strain. These changes in the levels of circadian clock transcription factors lead to changes in the muscle circadian transcriptome, which includes genes involved in metabolism, proteolysis, and xenobiotic detoxification. Moreover, a skeletal muscle-specific increase in expression extends lifespan and recapitulates some of the transcriptional and circadian changes that differentiate the long-lived from the parental strains. Altogether, these findings indicate that the muscle circadian clock is important for longevity and that circadian gene variants contribute to the evolutionary divergence in longevity across populations.
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http://dx.doi.org/10.1101/gr.246884.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6673717PMC
August 2019

Differentiation of human pluripotent stem cells into neurons or cortical organoids requires transcriptional co-regulation by UTX and 53BP1.

Nat Neurosci 2019 03 4;22(3):362-373. Epub 2019 Feb 4.

Department of Developmental Neurobiology and Division of Developmental Biology, St. Jude Children's Research Hospital, Memphis, TN, USA.

UTX is a chromatin modifier required for development and neural lineage specification, but how it controls these biological processes is unclear. To determine the molecular mechanisms of UTX, we identified novel UTX protein interaction partners. Here we show that UTX and 53BP1 directly interact and co-occupy promoters in human embryonic stem cells and differentiating neural progenitor cells. Human 53BP1 contains a UTX-binding site that diverges from its mouse homolog by 41%, and disruption of the 53BP1-UTX interaction abrogated human, but not mouse, neurogenesis in vitro. The 53BP1-UTX interaction is required to upregulate key neurodevelopmental genes during the differentiation of human embryonic stem cells into neurons or into cortical organoids. 53BP1 promotes UTX chromatin binding, and in turn H3K27 modifications and gene activation, at a subset of genomic regions, including neurogenic genes. Overall, our data suggest that the 53BP1-UTX interaction supports the activation of key genes required for human neurodevelopment.
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http://dx.doi.org/10.1038/s41593-018-0328-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6511450PMC
March 2019

Spectral Library Search Improves Assignment of TMT Labeled MS/MS Spectra.

J Proteome Res 2018 09 16;17(9):3325-3331. Epub 2018 Aug 16.

Department of Computer Science , University of Waterloo , Waterloo , Ontario N2L 3G1 , Canada.

Tandem mass tag (TMT)-based liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a proven approach for large-scale multiplexed protein quantification. However, the identification of TMT-labeled peptides is compromised by the labeling during traditional sequence database searches. In this study, we aim to use a spectral library search to increase the sensitivity and specificity of peptide identification for TMT-based MS data. Compared to MS/MS spectra of unlabeled peptides, the spectra of TMT-labeled counterparts usually display intensified b ions, suggesting that TMT labeling can alter product ion patterns during MS/MS fragementation. We compiled a human TMT spectral library of 401,168 unique peptides of high quality from millions of peptide-spectrum matches in tens of profiling projects, matching to 14,048 nonredundant proteins (13,953 genes). A mouse TMT spectral library of similar size was also constructed. The libraries were subsequently appended with decoy spectra to evaluate the false discovery rate, which was validated by a simulated null TMT data set. The performance of the library search was further optimized by removing TMT reporter ions and selecting an appropriate library construction method. Finally, we searched a human TMT data set against the spectral library to demonstrate that the spectral library outperformed the sequence database. Both human and mouse TMT libraries were made publicly available to the research community.
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http://dx.doi.org/10.1021/acs.jproteome.8b00594DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6390480PMC
September 2018

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification.

J Vis Exp 2017 11 15(129). Epub 2017 Nov 15.

St. Jude Proteomics Facility, St. Jude Children's Research Hospital; Departments of Structural Biology and Developmental Neurobiology, St. Jude Children's Research Hospital;

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) coupled to tandem mass spectrometry (LC-MS/MS) and isobaric labeling multiplexing capacity. Here, we introduce a deep-proteomics profiling protocol that combines 10-plex tandem mass tag (TMT) labeling with an extensive LC/LC-MS/MS platform, and post-MS computational interference correction to accurately quantitate whole proteomes. This protocol includes the following main steps: protein extraction and digestion, TMT labeling, 2-dimensional (2D) LC, high-resolution mass spectrometry, and computational data processing. Quality control steps are included for troubleshooting and evaluating experimental variation. More than 10,000 proteins in mammalian samples can be confidently quantitated with this protocol. This protocol can also be applied to the quantitation of post translational modifications with minor changes. This multiplexed, robust method provides a powerful tool for proteomic analysis in a variety of complex samples, including cell culture, animal tissues, and human clinical specimens.
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http://dx.doi.org/10.3791/56474DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5755397PMC
November 2017

Blocking an N-terminal acetylation-dependent protein interaction inhibits an E3 ligase.

Nat Chem Biol 2017 Aug 5;13(8):850-857. Epub 2017 Jun 5.

Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.

N-terminal acetylation is an abundant modification influencing protein functions. Because ∼80% of mammalian cytosolic proteins are N-terminally acetylated, this modification is potentially an untapped target for chemical control of their functions. Structural studies have revealed that, like lysine acetylation, N-terminal acetylation converts a positively charged amine into a hydrophobic handle that mediates protein interactions; hence, this modification may be a druggable target. We report the development of chemical probes targeting the N-terminal acetylation-dependent interaction between an E2 conjugating enzyme (UBE2M or UBC12) and DCN1 (DCUN1D1), a subunit of a multiprotein E3 ligase for the ubiquitin-like protein NEDD8. The inhibitors are highly selective with respect to other protein acetyl-amide-binding sites, inhibit NEDD8 ligation in vitro and in cells, and suppress anchorage-independent growth of a cell line with DCN1 amplification. Overall, our data demonstrate that N-terminal acetyl-dependent protein interactions are druggable targets and provide insights into targeting multiprotein E2-E3 ligases.
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http://dx.doi.org/10.1038/nchembio.2386DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5577376PMC
August 2017

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

Anal Chem 2017 03 22;89(5):2956-2963. Epub 2017 Feb 22.

Heilongjiang University of Chinese Medicine , Harbin, Heilongjiang 150040, China.

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 y 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 y 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.
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http://dx.doi.org/10.1021/acs.analchem.6b04415DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5467445PMC
March 2017

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

Cancer Cell 2015 Sep 27;28(3):343-56. Epub 2015 Aug 27.

Princess Margaret Cancer Centre, University Health Network, Toronto, ON M5G 2C4, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON M5G 1L7, Canada.

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.
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http://dx.doi.org/10.1016/j.ccell.2015.07.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4573904PMC
September 2015

Sequential Elution Interactome Analysis of the Mind Bomb 1 Ubiquitin Ligase Reveals a Novel Role in Dendritic Spine Outgrowth.

Mol Cell Proteomics 2015 Jul 30;14(7):1898-910. Epub 2015 Apr 30.

From the ‡Departments of Structural Biology and Developmental Neurobiology, §St. Jude Proteomics Facility,

The mind bomb 1 (Mib1) ubiquitin ligase is essential for controlling metazoan development by Notch signaling and possibly the Wnt pathway. It is also expressed in postmitotic neurons and regulates neuronal morphogenesis and synaptic activity by mechanisms that are largely unknown. We sought to comprehensively characterize the Mib1 interactome and study its potential function in neuron development utilizing a novel sequential elution strategy for affinity purification, in which Mib1 binding proteins were eluted under different stringency and then quantified by the isobaric labeling method. The strategy identified the Mib1 interactome with both deep coverage and the ability to distinguish high-affinity partners from low-affinity partners. A total of 817 proteins were identified during the Mib1 affinity purification, including 56 high-affinity partners and 335 low-affinity partners, whereas the remaining 426 proteins are likely copurified contaminants or extremely weak binding proteins. The analysis detected all previously known Mib1-interacting proteins and revealed a large number of novel components involved in Notch and Wnt pathways, endocytosis and vesicle transport, the ubiquitin-proteasome system, cellular morphogenesis, and synaptic activities. Immunofluorescence studies further showed colocalization of Mib1 with five selected proteins: the Usp9x (FAM) deubiquitinating enzyme, alpha-, beta-, and delta-catenins, and CDKL5. Mutations of CDKL5 are associated with early infantile epileptic encephalopathy-2 (EIEE2), a severe form of mental retardation. We found that the expression of Mib1 down-regulated the protein level of CDKL5 by ubiquitination, and antagonized CDKL5 function during the formation of dendritic spines. Thus, the sequential elution strategy enables biochemical characterization of protein interactomes; and Mib1 analysis provides a comprehensive interactome for investigating its role in signaling networks and neuronal development.
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http://dx.doi.org/10.1074/mcp.M114.045898DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4587327PMC
July 2015

Quantitative protein analysis by mass spectrometry.

Methods Mol Biol 2015 ;1278:281-305

St. Jude Proteomics Facility, St. Jude Children's Research Hospital, Memphis, TN, 38105, USA.

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.
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http://dx.doi.org/10.1007/978-1-4939-2425-7_17DOI Listing
December 2015

CENP-A K124 Ubiquitylation Is Required for CENP-A Deposition at the Centromere.

Dev Cell 2015 Mar 26;32(5):589-603. Epub 2015 Feb 26.

Center for Childhood Cancer and Blood Diseases, The Research Institute at Nationwide Children's Hospital, 700 Children's Drive, Columbus, OH 43205, USA; Department of Pediatrics, College of Medicine, The Ohio State University, 700 Children's Drive, Columbus, OH 43205, USA. Electronic address:

CENP-A is a centromere-specific histone H3 variant that epigenetically determines centromere identity to ensure kinetochore assembly and proper chromosome segregation, but the precise mechanism of its specific localization within centromeric heterochromatin remains obscure. We have discovered that CUL4A-RBX1-COPS8 E3 ligase activity is required for CENP-A ubiquitylation on lysine 124 (K124) and CENP-A centromere localization. A mutation of CENP-A, K124R, reduces interaction with HJURP (a CENP-A-specific histone chaperone) and abrogates localization of CENP-A to the centromere. Addition of monoubiquitin is sufficient to restore CENP-A K124R to centromeres and the interaction with HJURP, indicating that "signaling" ubiquitylation is required for CENP-A loading at centromeres. The CUL4A-RBX1 complex is required for loading newly synthesized CENP-A and maintaining preassembled CENP-A at centromeres. Thus, CENP-A K124R ubiquitylation, mediated by the CUL4A-RBX1-COPS8 complex, is essential for CENP-A deposition at the centromere.
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http://dx.doi.org/10.1016/j.devcel.2015.01.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4374629PMC
March 2015

Integrated approaches for analyzing U1-70K cleavage in Alzheimer's disease.

J Proteome Res 2014 Nov 13;13(11):4526-34. Epub 2014 Jun 13.

Departments of Structural Biology and Developmental Neurobiology, ‡St. Jude Proteomics Facility, St. Jude Children's Research Hospital , Memphis, Tennessee 38105, United States.

The accumulation of pathologic protein fragments is common in neurodegenerative disorders. We have recently identified in Alzheimer's disease (AD) the aggregation of the U1-70K splicing factor and abnormal RNA processing. Here, we present that U1-70K can be cleaved into an N-terminal truncation (N40K) in ∼50% of AD cases, and the N40K abundance is inversely proportional to the total level of U1-70K. To map the cleavage site, we compared tryptic peptides of N40K and stable isotope labeled U1-70K by liquid chromatography-tandem mass spectrometry (MS), revealing that the proteolysis site is located in a highly repetitive and hydrophilic domain of U1-70K. We then adapted Western blotting to map the cleavage site in two steps: (i) mass spectrometric analysis revealing that U1-70K and N40K share the same N-termini and contain no major modifications; (ii) matching N40K with a series of six recombinant U1-70K truncations to define the cleavage site within a small region (Arg300 ± 6 residues). Finally, N40K expression led to substantial degeneration of rat primary hippocampal neurons. In summary, we combined multiple approaches to identify the U1-70K proteolytic site and found that the N40K fragment might contribute to neuronal toxicity in Alzheimer's disease.
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http://dx.doi.org/10.1021/pr5003593DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4227550PMC
November 2014

Exercise protects against MPTP-induced neurotoxicity in mice.

Brain Res 2010 Jun 29;1341:72-83. Epub 2010 Jan 29.

Department of Psychology, Rhodes College, Memphis, TN 38112, USA.

Exercise has been shown to be potently neuroprotective in several neurodegenerative models, including 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) model of Parkinson's disease (PD). In order to determine the critical duration of exercise necessary for DA neuroprotection, mice were allowed to run for either 1, 2 or 3months prior to treatment with saline or MPTP. Quantification of DA neurons in the SNpc show that mice allowed to run unrestricted for 1 or 2months lost significant numbers of neurons following MPTP administration as compared to saline treated mice; however, 3months of exercise provided complete protection against MPTP-induced neurotoxicity. To determine the critical intensity of exercise for DA neuroprotection, mice were restricted in their running to either 1/3 or 2/3 that of the full running group for 3months prior to treatment with saline or MPTP. Quantification of DA neurons in the SNpc show that mice whose running was restricted lost significant numbers of DA neurons due to MPTP toxicity; however, the 2/3 running group demonstrated partial protection. Neurochemical analyses of DA and its metabolites DOPAC and HVA show that exercise also functionally protects neurons from MPTP-induced neurotoxicity. Proteomic analysis of SN and STR tissues indicates that 3months of exercise induces changes in proteins related to energy regulation, cellular metabolism, the cytoskeleton, and intracellular signaling events. Taken together, these data indicate that exercise potently protects DA neurons from acute MPTP toxicity, suggesting that this simple lifestyle element may also confer significant protection against developing PD in humans.
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http://dx.doi.org/10.1016/j.brainres.2010.01.053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2884060PMC
June 2010

Proteomic studies of the intrinsically unstructured mammalian proteome.

J Proteome Res 2006 Oct;5(10):2839-48

Department of Structural Biology, St. Jude Children's Research Hospital, and Department of Molecular Sciences, University of Tennessee Health Sciences Center, Memphis, Tennessee 38163, USA.

Intrinsically unstructured proteins (IUPs) represent an important class of proteins primarily involved in cellular signaling and regulation. The aim of this study was to develop methodology for the enrichment and identification of IUPs. We show that heat treatment of NIH3T3 mouse fibroblast cell extracts at 98 degrees C selects for IUPs. The majority of these IUPs were cytosolic or nuclear proteins involved in cell signaling or regulation. These studies represent the first large-scale experimental investigation of the intrinsically unstructured mammalian proteome.
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http://dx.doi.org/10.1021/pr060328cDOI Listing
October 2006

Cellular localization of D-lactate dehydrogenase and NADH oxidase from Archaeoglobus fulgidus.

Archaea 2002 Sep;1(2):95-104

Department of Microbiology, Molecular Biology, and Biochemistry, University of Idaho, Moscow, ID 83844-3052, USA.

Members of the genus Archaeoglobus are hyperthermophilic sulfate reducers with an optimal growth temperature of 83 degrees C. Archaeoglobus fulgidus can utilize simple compounds including D-lactate, L-lactate and pyruvate as the sole substrate for carbon and electrons for dissimilatory sulfate reduction. Previously we showed that this organism makes a D-lactate dehydrogenase (Dld) that requires FAD and Zn2+ for activity. To determine the cellular location and topology of Dld and to identify proteins that interact with Dld, an antibody directed against Dld was prepared. Immunocytochemical studies using gold particle-coated secondary antibodies show that more than 85% of Dld is associated with the membrane. A truncated form of Dld was detected in immunoblots of whole cells treated with protease, showing that Dld is an integral membrane protein and that a significant portion of Dld, including part of the FAD-binding pocket, is outside the membrane facing the S-layer. The gene encoding Dld is part of an operon that includes noxA2, which encodes one of several NADH oxidases in A. fulgidus. Previous studies have shown that NoxA2 remains bound to Dld during purification. Thin sections of A. fulgidus probed simultaneously with antibodies against Dld and NoxA2 show that both proteins co-localized to the same sites in the membrane. Although these data show a tight interaction between NoxA2 and Dld, the role of NoxA2 in electron transport reactions is unknown. Rather, NoxA2 may protect proteins involved in electron transfer by reducing O2 to H2O2 or H2O.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2685561PMC
http://dx.doi.org/10.1155/2002/297264DOI Listing
September 2002

T cell epitope "hotspots" on the HIV Type 1 gp120 envelope protein overlap with tryptic fragments displayed by mass spectrometry.

AIDS Res Hum Retroviruses 2005 Feb;21(2):165-70

Department of Immunology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.

Our previous work has shown that immunodominant T-helper cell epitopes cluster within distinct fragments on a single face of the HIV envelope gp120 protein. We show in this report that the general positions of immunodominant epitopes are shared by T cells derived from BALB/c, C57BL/6, and CB6F1 mice, yet the precise peptides recognized by the responding T cell populations may differ. In addition, we find that gp120 peptides displayed by tryptic digestion and mass spectrometry of a purified HIV envelope protein share location with peptides defined as immunodominant T cell targets. Results are consistent with the suggestion that gp120 peptide location influences antigen processing, which, in turn, influences the specificity of immunodominant T cells.
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http://dx.doi.org/10.1089/aid.2005.21.165DOI Listing
February 2005

Identification of a multifunctional binding site on Ubc9p required for Smt3p conjugation.

J Biol Chem 2002 Dec 26;277(49):47938-45. Epub 2002 Sep 26.

Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.

Ubiquitin-like proteins (ub-lps) are conjugated by a conserved enzymatic pathway, involving ATP-dependent activation at the C terminus by an activating enzyme (E1) and formation of a thiolester intermediate with a conjugating enzyme (E2) prior to ligation to the target. Ubc9, the E2 for SUMO, synthesizes polymeric chains in the presence of its E1 and MgATP. To better understand conjugation of ub-lps, we have performed mutational analysis of Saccharomyces cerevisiae Ubc9p, which conjugates the SUMO family member Smt3p. We have identified Ubc9p surfaces involved in thiolester bond and Smt3p-Smt3p chain formation. The residues involved in thiolester bond formation map to a surface we show is the E1 binding site, and E2s for other ub-lps are likely to bind to their E1s at a homologous site. We also find that this same surface binds Smt3p. A mutation that impairs binding to E1 but not Smt3p impairs thiolester bond formation, suggesting that it is the E1 interaction at this site that is crucial. Interestingly, other E2s and their relatives also use this same surface for binding to ubiquitin, E3s, and other proteins, revealing this to be a multipurpose binding site and suggesting that the entire E1-E2-E3 pathway has coevolved for a given ub-lp.
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http://dx.doi.org/10.1074/jbc.M207442200DOI Listing
December 2002
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