Publications by authors named "Junmin Peng"

193 Publications

JUMPt: Comprehensive Protein Turnover Modeling of In Vivo Pulse SILAC Data by Ordinary Differential Equations.

Anal Chem 2021 10 29;93(40):13495-13504. Epub 2021 Sep 29.

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

Recent advances in mass spectrometry (MS)-based proteomics allow the measurement of turnover rates of thousands of proteins using dynamic labeling methods, such as pulse stable isotope labeling by amino acids in cell culture (pSILAC). However, when applying the pSILAC strategy to multicellular animals (e.g., mice), the labeling process is significantly delayed by native amino acids recycled from protein degradation in vivo, raising a challenge of defining accurate protein turnover rates. Here, we report JUMPt, a software package using a novel ordinary differential equation (ODE)-based mathematical model to determine reliable rates of protein degradation. The uniqueness of JUMPt is to consider amino acid recycling and fit the kinetics of the labeling amino acid (e.g., Lys) and whole proteome simultaneously to derive half-lives of individual proteins. Multiple settings in the software are designed to enable simple to comprehensive data inputs for precise analysis of half-lives with flexibility. We examined the software by studying the turnover of thousands of proteins in the pSILAC brain and liver tissues. The results were largely consistent with the proteome turnover measurements from previous studies. The long-lived proteins are enriched in the integral membrane, myelin sheath, and mitochondrion in the brain. In summary, the ODE-based JUMPt software is an effective proteomics tool for analyzing large-scale protein turnover, and the software is publicly available on GitHub (https://github.com/JUMPSuite/JUMPt) to the research community.
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http://dx.doi.org/10.1021/acs.analchem.1c02309DOI Listing
October 2021

Acute depletion of CTCF rewires genome-wide chromatin accessibility.

Genome Biol 2021 08 24;22(1):244. Epub 2021 Aug 24.

Tumor Cell Biology, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN, 38105, USA.

Background: The transcription factor CTCF appears indispensable in defining topologically associated domain boundaries and maintaining chromatin loop structures within these domains, supported by numerous functional studies. However, acute depletion of CTCF globally reduces chromatin interactions but does not significantly alter transcription.

Results: Here, we systematically integrate multi-omics data including ATAC-seq, RNA-seq, WGBS, Hi-C, Cut&Run, and CRISPR-Cas9 survival dropout screens, and time-solved deep proteomic and phosphoproteomic analyses in cells carrying auxin-induced degron at endogenous CTCF locus. Acute CTCF protein degradation markedly rewires genome-wide chromatin accessibility. Increased accessible chromatin regions are frequently located adjacent to CTCF-binding sites at promoter regions and insulator sites associated with enhanced transcription of nearby genes. In addition, we use CTCF-associated multi-omics data to establish a combinatorial data analysis pipeline to discover CTCF co-regulatory partners. We successfully identify 40 candidates, including multiple established partners. Interestingly, many CTCF co-regulators that have alterations of their respective downstream gene expression do not show changes of their own expression levels across the multi-omics measurements upon acute CTCF loss, highlighting the strength of our system to discover hidden co-regulatory partners associated with CTCF-mediated transcription.

Conclusions: This study highlights that CTCF loss rewires genome-wide chromatin accessibility, which plays a critical role in transcriptional regulation.
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http://dx.doi.org/10.1186/s13059-021-02466-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8386078PMC
August 2021

Proteomic landscape of Alzheimer's Disease: novel insights into pathogenesis and biomarker discovery.

Mol Neurodegener 2021 08 12;16(1):55. Epub 2021 Aug 12.

Departments of Structural Biology and Developmental Neurobiology, St. Jude Children's Research Hospital, 38105, Memphis, TN, USA.

Mass spectrometry-based proteomics empowers deep profiling of proteome and protein posttranslational modifications (PTMs) in Alzheimer's disease (AD). Here we review the advances and limitations in historic and recent AD proteomic research. Complementary to genetic mapping, proteomic studies not only validate canonical amyloid and tau pathways, but also uncover novel components in broad protein networks, such as RNA splicing, development, immunity, membrane transport, lipid metabolism, synaptic function, and mitochondrial activity. Meta-analysis of seven deep datasets reveals 2,698 differentially expressed (DE) proteins in the landscape of AD brain proteome (n = 12,017 proteins/genes), covering 35 reported AD genes and risk loci. The DE proteins contain cellular markers enriched in neurons, microglia, astrocytes, oligodendrocytes, and epithelial cells, supporting the involvement of diverse cell types in AD pathology. We discuss the hypothesized protective or detrimental roles of selected DE proteins, emphasizing top proteins in "amyloidome" (all biomolecules in amyloid plaques) and disease progression. Comprehensive PTM analysis represents another layer of molecular events in AD. In particular, tau PTMs are correlated with disease stages and indicate the heterogeneity of individual AD patients. Moreover, the unprecedented proteomic coverage of biofluids, such as cerebrospinal fluid and serum, procures novel putative AD biomarkers through meta-analysis. Thus, proteomics-driven systems biology presents a new frontier to link genotype, proteotype, and phenotype, accelerating the development of improved AD models and treatment strategies.
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http://dx.doi.org/10.1186/s13024-021-00474-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8359598PMC
August 2021

Global Proteomic Profiling of Pediatric AML: A Pilot Study.

Cancers (Basel) 2021 Jun 24;13(13). Epub 2021 Jun 24.

Department of Pharmacotherapy and Translational Research, Center for Pharmacogenomics, College of Pharmacy, University of Florida, Gainesville, FL 32610, USA.

Acute Myeloid Leukemia (AML) is a heterogeneous disease with several recurrent cytogenetic abnormalities. Despite genomics and transcriptomics profiling efforts to understand AML's heterogeneity, studies focused on the proteomic profiles associated with pediatric AML cytogenetic features remain limited. Furthermore, the majority of biological functions within cells are operated by proteins (i.e., enzymes) and most drugs target the proteome rather than the genome or transcriptome, thus, highlighting the significance of studying proteomics. Here, we present our results from a pilot study investigating global proteomic profiles of leukemic cells obtained at diagnosis from 16 pediatric AML patients using a robust TMT-LC/LC-MS/MS platform. The proteome profiles were compared among patients with or without core binding factor (CBF) translocation indicated by a t(8;21) or inv(16) cytogenetic abnormality, minimal residual disease status at the end of the first cycle of chemotherapy (MRD1), and in vitro chemosensitivity of leukemic cells to cytarabine (Ara-C LC50). Our results established proteomic differences between CBF and non-CBF AML subtypes, providing insights to AML subtypes physiology, and identified potential druggable proteome targets such as , , , , , , , (isoform 1 and 2), , , and
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http://dx.doi.org/10.3390/cancers13133161DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8268478PMC
June 2021

Network-based systems pharmacology reveals heterogeneity in LCK and BCL2 signaling and therapeutic sensitivity of T-cell acute lymphoblastic leukemia.

Nat Cancer 2021 Mar 21;2(3):284-299. Epub 2021 Jan 21.

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

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy, and novel therapeutics are much needed. Profiling patient leukemia' drug sensitivities , we discovered that 44.4% of childhood and 16.7% of adult T-ALL cases exquisitely respond to dasatinib. Applying network-based systems pharmacology analyses to examine signal circuitry, we identified preTCR-LCK activation as the driver of dasatinib sensitivity, and T-ALL-specific LCK dependency was confirmed in genome-wide CRISPR-Cas9 screens. Dasatinib-sensitive T-ALLs exhibited high BCL-XL and low BCL2 activity and venetoclax resistance. Discordant sensitivity of T-ALL to dasatinib and venetoclax is strongly correlated with T-cell differentiation, particularly with the dynamic shift in LCK vs. BCL2 activation. Finally, single-cell analysis identified leukemia heterogeneity in LCK and BCL2 signaling and T-cell maturation stage, consistent with dasatinib response. In conclusion, our results indicate that developmental arrest in T-ALL drives differential activation of preTCR-LCK and BCL2 signaling in this leukemia, providing unique opportunities for targeted therapy.
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http://dx.doi.org/10.1038/s43018-020-00167-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8208590PMC
March 2021

Structural analysis of the full-length human LRRK2.

Cell 2021 Jun 8;184(13):3519-3527.e10. Epub 2021 Jun 8.

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

Mutations in leucine-rich repeat kinase 2 (LRRK2) are commonly implicated in the pathogenesis of both familial and sporadic Parkinson's disease (PD). LRRK2 regulates critical cellular processes at membranous organelles and forms microtubule-based pathogenic filaments, yet the molecular basis underlying these biological roles of LRRK2 remains largely enigmatic. Here, we determined high-resolution structures of full-length human LRRK2, revealing its architecture and key interdomain scaffolding elements for rationalizing disease-causing mutations. The kinase domain of LRRK2 is captured in an inactive state, a conformation also adopted by the most common PD-associated mutation, LRRK2. This conformation serves as a framework for structure-guided design of conformational specific inhibitors. We further determined the structure of COR-mediated LRRK2 dimers and found that single-point mutations at the dimer interface abolished pathogenic filamentation in cells. Overall, our study provides mechanistic insights into physiological and pathological roles of LRRK2 and establishes a structural template for future therapeutic intervention in PD.
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http://dx.doi.org/10.1016/j.cell.2021.05.004DOI Listing
June 2021

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

J Med Chem 2021 06 27;64(11):7296-7311. Epub 2021 May 27.

Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, United States.

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 (SJ6986) as a valuable chemical probe for studying the role of GSPT1/2 and , and it supports the utility of a diverse library of CRBN binders in the pursuit of targeting undruggable oncoproteins.
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http://dx.doi.org/10.1021/acs.jmedchem.0c01313DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8201443PMC
June 2021

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
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8495913PMC
May 2021

High-Throughput Profiling of Proteome and Posttranslational Modifications by 16-Plex TMT Labeling and Mass Spectrometry.

Methods Mol Biol 2021 ;2228:205-224

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

Mass spectrometry (MS)-based proteomic profiling of whole proteome and protein posttranslational modifications (PTMs) is a powerful technology to measure the dynamics of proteome with high throughput and deep coverage. The reproducibility of quantification benefits not only from the fascinating developments in high-performance liquid chromatography (LC) and high-resolution MS with enhanced scan rates but also from the invention of multiplexed isotopic labeling strategies, such as the tandem mass tags (TMT). In this chapter, we introduce a 16-plex TMT-LC/LC-MS/MS protocol for proteomic profiling of biological and clinical samples. The protocol includes protein extraction, enzymatic digestion, PTM peptide enrichment, TMT labeling, and two-dimensional reverse-phase liquid chromatography fractionation coupled with tandem mass spectrometry (MS/MS) analysis, followed by computational data processing. In general, more than 10,000 proteins and tens of thousands of PTM sites (e.g., phosphorylation and ubiquitination) can be confidently quantified. This protocol provides a general protein measurement tool, enabling the dissection of protein dysregulation in any biological samples and human diseases.
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http://dx.doi.org/10.1007/978-1-0716-1024-4_15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8458009PMC
June 2021

Quantifying Proteome and Protein Modifications in Activated T Cells by Multiplexed Isobaric Labeling Mass Spectrometry.

Methods Mol Biol 2021 ;2285:297-317

Center for Proteomics and Metabolomics, St. Jude Children's Research Hospital, Memphis, TN, USA.

The dynamic regulation of protein function by altered protein expression and post-translational modifications (PTMs) is essential for T cell function, but it has remained difficult to systemically quantify such events. Mass spectrometry (MS)-based proteomics has become a mainstream tool for comprehensive profiling of proteome and PTMs, especially with the development of multiplexed isobaric labeling methods, such as tandem mass tag (TMT), coupled with high-resolution two-dimensional liquid chromatography and tandem mass spectrometry (LC/LC-MS/MS). Here, we introduce a deep proteomics profiling protocol with an optimized 11-plex TMT-LC/LC-MS/MS platform to quantitate whole proteome, phosphoproteome, acetylome, and methylome in activated T cells. The major steps include preparation of activated T cells, protein extraction and digestion, TMT labeling, basic pH reverse phase LC, modified peptide enrichment, acidic pH reverse phase LC-MS/MS, and computational data processing. Approximately 10,000 proteins, 30,000 phosphosites, 2,000 lysine acetylated sites, and 1,000 lysine methylated sites can be identified and quantified from 1 mg of proteins per sample. Quality control steps are implemented in this protocol, and future development, such as nanoscale 16-plex TMT analysis, is discussed. This multiplexed and robust method provides a powerful tool for dissecting proteomic and PTM signatures in T cells at the systems level, and it is equally suitable for other biological samples, including effector T cell subsets.
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http://dx.doi.org/10.1007/978-1-0716-1311-5_23DOI Listing
June 2021

Global Profiling of Lysine Accessibility to Evaluate Protein Structure Changes in Alzheimer's Disease.

J Am Soc Mass Spectrom 2021 Apr 8;32(4):936-945. Epub 2021 Mar 8.

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

The linear sequence of amino acids in a protein folds into a 3D structure to execute protein activity and function, but it is still challenging to profile the 3D structure at the proteome scale. Here, we present a method of native protein tandem mass tag (TMT) profiling of Lys accessibility and its application to investigate structural alterations in human brain specimens of Alzheimer's disease (AD). In this method, proteins are extracted under a native condition, labeled by TMT reagents, followed by trypsin digestion and peptide analysis using two-dimensional liquid chromatography and tandem mass spectrometry (LC/LC-MS/MS). The method quantifies Lys labeling efficiency to evaluate its accessibility on the protein surface, which may be affected by protein conformations, protein modifications, and/or other molecular interactions. We systematically optimized the amount of TMT reagents, reaction time, and temperature and then analyzed protein samples under multiple conditions, including different labeling time (5 and 30 min), heat treatment, AD and normal human cases. The experiment profiled 15370 TMT-labeled peptides in 4475 proteins. As expected, the heat treatment led to extensive changes in protein conformations, with 17% of the detected proteome displaying differential labeling. Compared to the normal controls, AD brain showed different Lys accessibility of tau and RNA splicing complexes, which are the hallmarks of AD pathology, as well as proteins involved in transcription, mitochondrial, and synaptic functions. To eliminate the possibility that the observed differential Lys labeling was caused by protein level change, the whole proteome was quantified with standard TMT-LC/LC-MS/MS for normalization. Thus, this native protein TMT method enables the proteome-wide measurement of Lys accessibility, representing a straightforward strategy to explore protein structure in any biological system.
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http://dx.doi.org/10.1021/jasms.0c00450DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8255072PMC
April 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

FBXO11-mediated proteolysis of BAHD1 relieves PRC2-dependent transcriptional repression in erythropoiesis.

Blood 2021 01;137(2):155-167

Department of Hematology.

The histone mark H3K27me3 and its reader/writer polycomb repressive complex 2 (PRC2) mediate widespread transcriptional repression in stem and progenitor cells. Mechanisms that regulate this activity are critical for hematopoietic development but are poorly understood. Here we show that the E3 ubiquitin ligase F-box only protein 11 (FBXO11) relieves PRC2-mediated repression during erythroid maturation by targeting its newly identified substrate bromo adjacent homology domain-containing 1 (BAHD1), an H3K27me3 reader that recruits transcriptional corepressors. Erythroblasts lacking FBXO11 are developmentally delayed, with reduced expression of maturation-associated genes, most of which harbor bivalent histone marks at their promoters. In FBXO11-/- erythroblasts, these gene promoters bind BAHD1 and fail to recruit the erythroid transcription factor GATA1. The BAHD1 complex interacts physically with PRC2, and depletion of either component restores FBXO11-deficient erythroid gene expression. Our studies identify BAHD1 as a novel effector of PRC2-mediated repression and reveal how a single E3 ubiquitin ligase eliminates PRC2 repression at many developmentally poised bivalent genes during erythropoiesis.
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http://dx.doi.org/10.1182/blood.2020007809DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7820877PMC
January 2021

Combinatorial expression of GPCR isoforms affects signalling and drug responses.

Nature 2020 11 4;587(7835):650-656. Epub 2020 Nov 4.

MRC Laboratory of Molecular Biology, Cambridge, UK.

G-protein-coupled receptors (GPCRs) are membrane proteins that modulate physiology across human tissues in response to extracellular signals. GPCR-mediated signalling can differ because of changes in the sequence or expression of the receptors, leading to signalling bias when comparing diverse physiological systems. An underexplored source of such bias is the generation of functionally diverse GPCR isoforms with different patterns of expression across different tissues. Here we integrate data from human tissue-level transcriptomes, GPCR sequences and structures, proteomics, single-cell transcriptomics, population-wide genetic association studies and pharmacological experiments. We show how a single GPCR gene can diversify into several isoforms with distinct signalling properties, and how unique isoform combinations expressed in different tissues can generate distinct signalling states. Depending on their structural changes and expression patterns, some of the detected isoforms may influence cellular responses to drugs and represent new targets for developing drugs with improved tissue selectivity. Our findings highlight the need to move from a canonical to a context-specific view of GPCR signalling that considers how combinatorial expression of isoforms in a particular cell type, tissue or organism collectively influences receptor signalling and drug responses.
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http://dx.doi.org/10.1038/s41586-020-2888-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7611127PMC
November 2020

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

Integrated analysis of ultra-deep proteomes in cortex, cerebrospinal fluid and serum reveals a mitochondrial signature in Alzheimer's disease.

Mol Neurodegener 2020 07 25;15(1):43. Epub 2020 Jul 25.

Departments of Structural Biology and Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, TN, 38105, USA.

Background: Based on amyloid cascade and tau hypotheses, protein biomarkers of different Aβ and tau species in cerebrospinal fluid (CSF) and blood/plasma/serum have been examined to correlate with brain pathology. Recently, unbiased proteomic profiling of these human samples has been initiated to identify a large number of novel AD biomarker candidates, but it is challenging to define reliable candidates for subsequent large-scale validation.

Methods: We present a comprehensive strategy to identify biomarker candidates of high confidence by integrating multiple proteomes in AD, including cortex, CSF and serum. The proteomes were analyzed by the multiplexed tandem-mass-tag (TMT) method, extensive liquid chromatography (LC) fractionation and high-resolution tandem mass spectrometry (MS/MS) for ultra-deep coverage. A systems biology approach was used to prioritize the most promising AD signature proteins from all proteomic datasets. Finally, candidate biomarkers identified by the MS discovery were validated by the enzyme-linked immunosorbent (ELISA) and TOMAHAQ targeted MS assays.

Results: We quantified 13,833, 5941, and 4826 proteins from human cortex, CSF and serum, respectively. Compared to other studies, we analyzed a total of 10 proteomic datasets, covering 17,541 proteins (13,216 genes) in 365 AD, mild cognitive impairment (MCI) and control cases. Our ultra-deep CSF profiling of 20 cases uncovered the majority of previously reported AD biomarker candidates, most of which, however, displayed no statistical significance except SMOC1 and TGFB2. Interestingly, the AD CSF showed evident decrease of a large number of mitochondria proteins that were only detectable in our ultra-deep analysis. Further integration of 4 cortex and 4 CSF cohort proteomes highlighted 6 CSF biomarkers (SMOC1, C1QTNF5, OLFML3, SLIT2, SPON1, and GPNMB) that were consistently identified in at least 2 independent datasets. We also profiled CSF in the 5xFAD mouse model to validate amyloidosis-induced changes, and found consistent mitochondrial decreases (SOD2, PRDX3, ALDH6A1, ETFB, HADHA, and CYB5R3) in both human and mouse samples. In addition, comparison of cortex and serum led to an AD-correlated protein panel of CTHRC1, GFAP and OLFM3. In summary, 37 proteins emerged as potential AD signatures across cortex, CSF and serum, and strikingly, 59% of these were mitochondria proteins, emphasizing mitochondrial dysfunction in AD. Selected biomarker candidates were further validated by ELISA and TOMAHAQ assays. Finally, we prioritized the most promising AD signature proteins including SMOC1, TAU, GFAP, SUCLG2, PRDX3, and NTN1 by integrating all proteomic datasets.

Conclusions: Our results demonstrate that novel AD biomarker candidates are identified and confirmed by proteomic studies of brain tissue and biofluids, providing a rich resource for large-scale biomarker validation for the AD community.
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http://dx.doi.org/10.1186/s13024-020-00384-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7382148PMC
July 2020

Toxicoproteomic Profiling of Transgenic Mice Treated with Rifampicin and Isoniazid.

Cells 2020 07 9;9(7). Epub 2020 Jul 9.

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

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 -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 B deficiency), peripheral neuropathy, and vitamin B 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 and mice treated with combinations of rifampicin and isoniazid. Proteomic profiling analysis suggested that the 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 B metabolism, and vitamin B 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.
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http://dx.doi.org/10.3390/cells9071654DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7407182PMC
July 2020

Control of Early B Cell Development by the RNA N-Methyladenosine Methylation.

Cell Rep 2020 06;31(13):107819

Department of Medicine, Section of Rheumatology and Gwen Knapp Center for Lupus and Immunology Research, The University of Chicago, Chicago, IL 60637, USA; Committee on Immunology, The University of Chicago, Chicago, IL 60637, USA. Electronic address:

The RNA N-methyladenosine (mA) methylation is installed by the METTL3-METTL14 methyltransferase complex. This modification has critical regulatory roles in various biological processes. Here, we report that deletion of Mettl14 dramatically reduces mRNA mA methylation in developing B cells and severely blocks B cell development in mice. Deletion of Mettl14 impairs interleukin-7 (IL-7)-induced pro-B cell proliferation and the large-pre-B-to-small-pre-B transition and causes dramatic abnormalities in gene expression programs important for B cell development. Suppression of a group of transcripts by cytoplasmic mA reader YTHDF2 is critical to the IL-7-induced pro-B cell proliferation. In contrast, the block in the large-pre-B-to-small-pre-B transition is independent of YTHDF1 or YTHDF2 but is associated with a failure to properly upregulate key transcription factors regulating this transition. Our data highlight the important regulatory roles of the RNA mA methylation and its reader proteins in early B cell development.
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http://dx.doi.org/10.1016/j.celrep.2020.107819DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7371152PMC
June 2020

JUMPm: A Tool for Large-Scale Identification of Metabolites in Untargeted Metabolomics.

Metabolites 2020 May 12;10(5). Epub 2020 May 12.

Center for Proteomics and Metabolomics, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.

Metabolomics is increasingly important for biomedical research, but large-scale metabolite identification in untargeted metabolomics is still challenging. Here, we present Jumbo Mass spectrometry-based Program of Metabolomics (JUMPm) software, a streamlined software tool for identifying potential metabolite formulas and structures in mass spectrometry. During database search, the false discovery rate is evaluated by a target-decoy strategy, where the decoys are produced by breaking the octet rule of chemistry. We illustrated the utility of JUMPm by detecting metabolite formulas and structures from liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) analyses of unlabeled and stable-isotope labeled yeast samples. We also benchmarked the performance of JUMPm by analyzing a mixed sample from a commercially available metabolite library in both hydrophilic and hydrophobic LC-MS/MS. These analyses confirm that metabolite identification can be significantly improved by estimating the element composition in formulas using stable isotope labeling, or by introducing LC retention time during a spectral library search, which are incorporated into JUMPm functions. Finally, we compared the performance of JUMPm and two commonly used programs, Compound Discoverer 3.1 and MZmine 2, with respect to putative metabolite identifications. Our results indicate that JUMPm is an effective tool for metabolite identification of both unlabeled and labeled data in untargeted metabolomics.
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http://dx.doi.org/10.3390/metabo10050190DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7281133PMC
May 2020

Ligand-induced monoubiquitination of BIK1 regulates plant immunity.

Nature 2020 05 22;581(7807):199-203. Epub 2020 Apr 22.

Institute for Plant Genomics and Biotechnology, Texas A&M University, College Station, TX, USA.

Recognition of microbe-associated molecular patterns (MAMPs) by pattern recognition receptors (PRRs) triggers the first line of inducible defence against invading pathogens. Receptor-like cytoplasmic kinases (RLCKs) are convergent regulators that associate with multiple PRRs in plants. The mechanisms that underlie the activation of RLCKs are unclear. Here we show that when MAMPs are detected, the RLCK BOTRYTIS-INDUCED KINASE 1 (BIK1) is monoubiquitinated following phosphorylation, then released from the flagellin receptor FLAGELLIN SENSING 2 (FLS2)-BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1) complex, and internalized dynamically into endocytic compartments. The Arabidopsis E3 ubiquitin ligases RING-H2 FINGER A3A (RHA3A) and RHA3B mediate the monoubiquitination of BIK1, which is essential for the subsequent release of BIK1 from the FLS2-BAK1 complex and activation of immune signalling. Ligand-induced monoubiquitination and endosomal puncta of BIK1 exhibit spatial and temporal dynamics that are distinct from those of the PRR FLS2. Our study reveals the intertwined regulation of PRR-RLCK complex activation by protein phosphorylation and ubiquitination, and shows that ligand-induced monoubiquitination contributes to the release of BIK1 family RLCKs from the PRR complex and activation of PRR signalling.
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http://dx.doi.org/10.1038/s41586-020-2210-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7233372PMC
May 2020

27-Plex Tandem Mass Tag Mass Spectrometry for Profiling Brain Proteome in Alzheimer's Disease.

Anal Chem 2020 05 7;92(10):7162-7170. Epub 2020 May 7.

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

Multiplexed isobaric labeling methods, such as tandem mass tags (TMT), remarkably improve the throughput of quantitative mass spectrometry. Here, we present a 27-plex TMT method coupled with two-dimensional liquid chromatography (LC/LC) for extensive peptide fractionation and high-resolution tandem mass spectrometry (MS/MS) for peptide quantification and then apply the method to profile the complex human brain proteome of Alzheimer's disease (AD). The 27-plex method combines multiplexed capacities of the 11-plex and the 16-plex TMT, as the peptides labeled by the two TMT sets display different mass and hydrophobicity, which can be well separated in LC-MS/MS. We first systematically optimized the protocol for the newly developed 16-plex TMT, including labeling reaction, desalting, and MS conditions, and then directly compared the 11-plex and 16-plex methods by analyzing the same human AD samples. Both methods yielded similar proteome coverage, analyzing >100 000 peptides in >10 000 human proteins. Furthermore, the 11-plex and 16-plex samples were mixed for a 27-plex assay, resulting in more than 8000 protein measurements within the same MS time. The 27-plex results are highly consistent with those of the individual 11-plex and 16-plex TMT analyses. We also used these proteomics data sets to compare the AD brain with the nondementia controls, discovering major AD-related proteins and revealing numerous novel protein alterations enriched in the pathways of amyloidosis, immunity, mitochondrial, and synaptic functions. Overall, our data strongly demonstrate that this new 27-plex strategy is highly feasible for routine large-scale proteomic analysis.
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http://dx.doi.org/10.1021/acs.analchem.0c00655DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8176402PMC
May 2020

Novel specialized cell state and spatial compartments within the germinal center.

Nat Immunol 2020 06 27;21(6):660-670. Epub 2020 Apr 27.

Department of Medicine, Section of Rheumatology and Gwen Knapp Center for Lupus and Immunology Research, University of Chicago, Chicago, IL, USA.

Within germinal centers (GCs), complex and highly orchestrated molecular programs must balance proliferation, somatic hypermutation and selection to both provide effective humoral immunity and to protect against genomic instability and neoplastic transformation. In contrast to this complexity, GC B cells are canonically divided into two principal populations, dark zone (DZ) and light zone (LZ) cells. We now demonstrate that, following selection in the LZ, B cells migrated to specialized sites within the canonical DZ that contained tingible body macrophages and were sites of ongoing cell division. Proliferating DZ (DZp) cells then transited into the larger DZ to become differentiating DZ (DZd) cells before re-entering the LZ. Multidimensional analysis revealed distinct molecular programs in each population commensurate with observed compartmentalization of noncompatible functions. These data provide a new three-cell population model that both orders critical GC functions and reveals essential molecular programs of humoral adaptive immunity.
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http://dx.doi.org/10.1038/s41590-020-0660-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7255947PMC
June 2020

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

Mol Cell 2020 03 21;77(6):1206-1221.e7. Epub 2020 Jan 21.

Department of Cell and Molecular Biology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.molcel.2019.12.022DOI Listing
March 2020

An ABC Transporter Drives Medulloblastoma Pathogenesis by Regulating Sonic Hedgehog Signaling.

Cancer Res 2020 04 16;80(7):1524-1537. Epub 2020 Jan 16.

Department of Pharmaceutical Sciences, St. Jude Children's Research Hospital, Memphis, Tennessee.

Mutations in Sonic hedgehog (SHH) signaling promote aberrant proliferation and tumor growth. SHH-medulloblastoma (MB) is among the most frequent brain tumors in children less than 3 years of age. Although key components of the SHH pathway are well-known, we hypothesized that new disease-modifying targets of SHH-MB might be identified from large-scale bioinformatics and systems biology analyses. Using a data-driven systems biology approach, we built a MB-specific interactome. The ATP-binding cassette transporter ABCC4 was identified as a modulator of SHH-MB. Accordingly, increased expression correlated with poor overall survival in patients with SHH-MB. Knockdown of ABCC4 expression markedly blunted the constitutive activation of the SHH pathway secondary to or insufficiency. In human tumor cell lines, ABCC4 knockdown and inhibition reduced full-length GLI3 levels. In a clinically relevant murine SHH-MB model, targeted ablation of in primary tumors significantly reduced tumor burden and extended the lifespan of tumor-bearing mice. These studies reveal ABCC4 as a potent SHH pathway regulator and a new candidate to target with the potential to improve SHH-MB therapy. SIGNIFICANCE: These findings identify ABCC4 transporter as a new target in SHH-MB, prompting the development of inhibitors or the repurporsing of existing drugs to target ABCC4.
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http://dx.doi.org/10.1158/0008-5472.CAN-19-2054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7416697PMC
April 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

Cerebrospinal fluid tau fragment correlates with tau PET: a candidate biomarker for tangle pathology.

Brain 2020 02;143(2):650-660

Institute of Neuroscience and Physiology, Department of Psychiatry and Neurochemistry, the Sahlgrenska Academy at the University of Gothenburg, Mölndal, Sweden.

To date, there is no validated fluid biomarker for tau pathology in Alzheimer's disease, with contradictory results from studies evaluating the correlation between phosphorylated tau in CSF with tau PET imaging. Tau protein is subjected to proteolytic processing into fragments before being secreted to the CSF. A recent study suggested that tau cleavage after amino acid 368 by asparagine endopeptidase (AEP) is upregulated in Alzheimer's disease. We used immunoprecipitation followed by mass spectrometric analyses to evaluate the presence of tau368 species in CSF. A novel Simoa® assay for quantification of tau368 in CSF was developed, while total tau (t-tau) was measured by ELISA and the presence of tau368 in tangles was evaluated using immunohistochemistry. The diagnostic utility of tau368 was first evaluated in a pilot study (Alzheimer's disease = 20, control = 20), then in a second cohort where the IWG-2 biomarker criteria were applied (Alzheimer's disease = 37, control = 45), and finally in a third cohort where the correlation with 18F-GTP1 tau PET was evaluated (Alzheimer's disease = 38, control = 11). The tau368/t-tau ratio was significantly decreased in Alzheimer's disease (P < 0.001) in all cohorts. Immunohistochemical staining demonstrated that tau fragments ending at 368 are present in tangles. There was a strong negative correlation between the CSF tau368/t-tau ratio and 18F-GTP1 retention. Our data suggest that tau368 is a tangle-enriched fragment and that the CSF ratio tau368/t-tau reflects tangle pathology. This novel tau biomarker could be used to improve diagnosis of Alzheimer's disease and to facilitate the development of drug candidates targeting tau pathology. Furthermore, future longitudinal studies will increase our understanding of tau pathophysiology in Alzheimer's disease and other tauopathies.
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http://dx.doi.org/10.1093/brain/awz346DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7009597PMC
February 2020
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