Stable Isotope Labeling Of Amino Acids In Cell Culture Research Articles

TEX13B is essential for metabolic reprogramming during germ cell differentiation.

What is the functional significance of Tex13b in male germ cell development and differentiation? Tex13b regulates male germ cell differentiation by metabolic reprogramming during spermatogenesis. Studies in mice and humans suggest that TEX13B is a transcription factor and is exclusively expressed in germ cells. We sequenced the coding regions of TEX13B in 628 infertile men and 427 ethnically matched fertile control men. Further, to identify the molecular function of Tex13b, we created a Tex13b knockout and conditional overexpression system in GC-1spg (hereafter, GC-1) cells. Our recent exome sequencing study identified novel candidate genes for male infertility. TEX13B was found to be one of the potential candidates, hence we explored the role of TEX13B in male infertility within a large infertile case-control cohort. We performed functional analyses of Tex13b in a GC-1 cell line using CRISPR-Cas9. We differentially labelled the cell proteins by stable isotope labelling of amino acids in cell culture (SILAC) and performed mass spectrometry-based whole-cell proteomics to identify the differential protein regulation in knockout cells compared to wild-type cells. We found that Tex13b knockout leads to downregulation of the OXPHOS complexes and upregulation of glycolysis genes, which was further validated by western blotting. These results were further confirmed by respirometry analysis in Tex13b knockout cells. Further, we also performed a conditional overexpression of TEX13B in GC-1 cells and studied the expression of OXPHOS complex proteins by western blotting. We identified a rare variant, rs775429506 (p.Gly237Glu), exclusively in two non-obstructive-azoospermia (NOA) men, that may genetically predispose these men for infertility. Further, we demonstrated that Tex13b functions in the transcription regulation of OXPHOS complexes. N/A. We examined the function of Tex13b in GC-1 in vitro by knocking out and conditional overexpression, for understanding the function of Tex13b in germ cells. Unfortunately, this could not be replicated in either an animal model or in patient-derived tissue due to the non-availability of an animal model or patient's testis biopsies. This study identified that Tex13b plays an important role in male germ cell development and differentiation. The findings of this study would be useful for screening infertile males with spermatogenic failure and counselling them before the implementation of assisted reproduction technique(s). Funding was provided by the Council of Scientific and Industrial Research (CSIR) under the network project (BSC0101 and MLP0113) and SERB, the Department of Science and Technology, Government of India (J C Bose Fellowship: JCB/2019/000027). The authors do not have any competing interest.

High-Throughput Mass Spectrometry Analysis of N-Glycans and Protein Markers after FUT8 Knockdown in the Syngeneic SW480/SW620 Colorectal Cancer Cell Model.

Disruption of the glycosylation machinery is a common feature in many types of cancer, and colorectal cancer (CRC) is no exception. Core fucosylation is mediated by the enzyme fucosyltransferase 8 (FucT-8), which catalyzes the addition of α1,6-l-fucose to the innermost GlcNAc residue of N-glycans. We and others have documented the involvement of FucT-8 and core-fucosylated proteins in CRC progression, in which we addressed core fucosylation in the syngeneic CRC model formed by SW480 and SW620 tumor cell lines from the perspective of alterations in their N-glycosylation profile and protein expression as an effect of the knockdown of the FUT8 gene that encodes FucT-8. Using label-free, semiquantitative mass spectrometry (MS) analysis, we found noticeable differences in N-glycosylation patterns in FUT8-knockdown cells, affecting core fucosylation and sialylation, the Hex/HexNAc ratio, and antennarity. Furthermore, stable isotopic labeling of amino acids in cell culture (SILAC)-based proteomic screening detected the alteration of species involved in protein folding, endoplasmic reticulum (ER) and Golgi post-translational stabilization, epithelial polarity, and cellular response to damage and therapy. This data is available via ProteomeXchange with identifier PXD050012. Overall, the results obtained merit further investigation to validate their feasibility as biomarkers of progression and malignization in CRC, as well as their potential usefulness in clinical practice.

Involvement of the AKT Pathway in Resistance to Erlotinib and Cabozantinib in Triple-Negative Breast Cancer Cell Lines.

Resistance to protein tyrosine kinase inhibitors (TKIs) presents a significant challenge in therapeutic target development for cancers such as triple-negative breast cancer (TNBC), where conventional therapies are ineffective at combatting systemic disease. Due to increased expression, the receptor tyrosine kinases EGFR (epidermal growth factor receptor) and c-Met are potential targets for treatment. However, targeted anti-EGFR and anti-c-Met therapies have faced mixed results in clinical trials due to acquired resistance. We hypothesize that adaptive responses in regulatory kinase networks within the EGFR and c-Met signaling axes contribute to the development of acquired erlotinib and cabozantinib resistance. To test this, we developed two separate models for cabozantinib and erlotinib resistance using the MDA-MB-231 and MDA-MB-468 cell lines, respectively. We observed that erlotinib- or cabozantinib-resistant cell lines demonstrate enhanced cell proliferation, migration, invasion, and activation of EGFR or c-Met downstream signaling (respectively). Using a SILAC (Stable Isotope Labeling of Amino acids in Cell Culture)-labeled quantitative mass spectrometry proteomics approach, we assessed the effects of erlotinib or cabozantinib resistance on the phosphoproteome, proteome, and kinome. Using this integrated proteomics approach, we identified several potential kinase mediators of cabozantinib resistance and confirmed the contribution of AKT1 to erlotinib resistance in TNBC-resistant cell lines.

HyperSCP: Combining Isotopic and Isobaric Labeling for Higher Throughput Single-Cell Proteomics.

Recent developments in mass spectrometry-based single-cell proteomics (SCP) have resulted in dramatically improved sensitivity, yet the relatively low measurement throughput remains a limitation. Isobaric and isotopic labeling methods have been separately applied to SCP to increase throughput through multiplexing. Here we combined both forms of labeling to achieve multiplicative scaling for higher throughput. Two-plex stable isotope labeling of amino acids in cell culture (SILAC) and isobaric tandem mass tag (TMT) labeling enabled up to 28 single cells to be analyzed in a single liquid chromatography-mass spectrometry (LC-MS) analysis, in addition to carrier, reference, and negative control channels. A custom nested nanowell chip was used for nanoliter sample processing to minimize sample losses. Using a 145-min total LC-MS cycle time, ∼280 single cells were analyzed per day. This measurement throughput could be increased to ∼700 samples per day with a high-duty-cycle multicolumn LC system producing the same active gradient. The labeling efficiency and achievable proteome coverage were characterized for multiple analysis conditions.

Francisella tularensis Glyceraldehyde-3-Phosphate Dehydrogenase Is Relocalized during Intracellular Infection and Reveals Effect on Cytokine Gene Expression and Signaling.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is known for its multifunctionality in several pathogenic bacteria. Our previously reported data suggest that the GAPDH homologue of Francisella tularensis, GapA, might also be involved in other processes beyond metabolism. In the present study, we explored GapA's potential implication in pathogenic processes at the host cell level. Using immunoelectron microscopy, we demonstrated the localization of this bacterial protein inside infected macrophages and its peripheral distribution in bacterial cells increasing with infection time. A quantitative proteomic approach based on stable isotope labeling of amino acids in cell culture (SILAC) combined with pull-down assay enabled the identification of several of GapA's potential interacting partners within the host cell proteome. Two of these partners were further confirmed by alternative methods. We also investigated the impact of gapA deletion on the transcription of selected cytokine genes and the activation of the main signaling pathways. Our results show that ∆gapA-induced transcription of genes encoding several cytokines whose expressions were not affected in cells infected with a fully virulent wild-type strain. That might be caused, at least in part, by the detected differences in ERK/MAPK signaling activation. The experimental observations together demonstrate that the F. tularensis GAPDH homologue is directly implicated in multiple host cellular processes and, thereby, that it participates in several molecular mechanisms of pathogenesis.

Quantitative proteomic analysis reveals the effects of mu opioid agonists on HT22 cells.

Introduction: At present, the mu opioid receptor is the most important neuroaesthetics receptor in anesthesiology research, and the damage that it does to the nervous system is unknown. Methods: We investigated the effects of loperamide, an agonist of the mu opioid receptor, on protein expression in HT22 cells using stable isotope labeling of amino acids in cell culture (SILAC), immobilized metal affinity chromatography (IMAC) enrichment, and high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 7,823 proteins were identified. Results and Discussion: Bioinformatic analysis revealed that mu opioid receptor agonism can induce distinct changes in the proteome of HT22 cells. These findings improve our understanding of narcotic drugs.

Atypical E3 ligase ZFP91 promotes small-molecule-induced E2F2 transcription factor degradation for cancer therapy.

The E2F family of transcription factors play a crucial role in the development of various cancers. However, E2F members lack targetable binding pockets and are typically considered "undruggable". Unlike canonical small-molecule therapeutics, molecular glues mediate new E3 ligase-protein interactions to induce selective proteasomal degradation, which represents an attractive option to overcome these limitations. Human proteome microarray was utilized to identify a natural product-derived molecular glue for targeting E2F2 degradation. Co-IP analysis with stable isotope labeling of amino acids in cell culture (SILAC)-based quantitative proteomics was carried out to further explore the E3 ligase for E2F2 degradation. In this study, we identified a molecular glue bufalin, which significantly promoted E2F2 degradation. Unexpectedly, E2F2 underwent ubiquitination and proteasomal degradation via a previously undisclosed atypical E3 ligase, zinc finger protein 91 (ZFP91). In particular, we observed that bufalin markedly promoted E2F2-ZFP91 complex formation, thereby leading to E2F2 polyubiquitination via K48-linked ubiquitin chains for degradation. E2F2 degradation subsequently caused transcriptional suppression of multiple oncogenes including c-Myc, CCNE1, CCNE2, MCM5 and CDK1, and inhibited hepatocellular carcinoma growth invitro and invivo. Collectively, our findings open up a new direction for transcription factors degradation by targeting atypical E3 ligase ZFP91. Meanwhile, the chemical knockdown strategy with molecular glue may promote innovative transcription factor degrader development in cancer therapy. This work was financially supported by the NationalKeyResearchandDevelopment Projectof China (2022YFC3501601), National Natural Sciences Foundation of China (81973505, 82174008, 82030114), and China Postdoctoral Science Foundation (2019M650396), the Fundamental Research Funds for the Central Universities.

LOXL2-dependent deacetylation of aldolase A induces metabolic reprogramming and tumor progression

Lysyl-oxidase like-2 (LOXL2) regulates extracellular matrix remodeling and promotes tumor invasion and metastasis. Altered metabolism is a core hallmark of cancer, however, it remains unclear whether and how LOXL2 contributes to tumor metabolism. Here, we found that LOXL2 and its catalytically inactive L2Δ13 splice variant boost glucose metabolism of esophageal tumor cells, facilitate tumor cell proliferation and promote tumor development in vivo. Consistently, integrated transcriptomic and metabolomic analysis of a knock-in mouse model expressing L2Δ13 gene revealed that LOXL2/L2Δ13 overexpression perturbs glucose and lipid metabolism. Mechanistically, we identified aldolase A, glyceraldehyde-3-phosphate dehydrogenase and enolase as glycolytic proteins that interact physically with LOXL2 and L2Δ13. In the case of aldolase A, LOXL2/L2Δ13 stimulated its mobilization from the actin cytoskeleton to enhance aldolase activity during malignant transformation. Using stable isotope labeling of amino acids in cell culture (SILAC) followed by proteomic analysis, we identified LOXL2 and L2Δ13 as novel deacetylases that trigger metabolic reprogramming. Both LOXL2 and L2Δ13 directly catalyzed the deacetylation of aldolase A at K13, resulting in enhanced glycolysis which subsequently reprogramed tumor metabolism and promoted tumor progression. High level expression of LOXL2/L2Δ13 combined with decreased acetylation of aldolase-K13 predicted poor clinical outcome in patients with esophageal cancer. In summary, we have characterized a novel molecular mechanism that mediates the pro-tumorigenic activity of LOXL2 independently of its classical amine oxidase activity. These findings may enable the future development of therapeutic agents targeting the metabolic machinery via LOXL2 or L2Δ13. Highlight of the studyLOXL2 and its catalytically inactive isoform L2Δ13 function as new deacetylases to promote metabolic reprogramming and tumor progression in esophageal cancer by directly activating glycolytic enzymes such as aldolase A.

Characterization of RARRES1 Expression on Circulating Tumor Cells as Unfavorable Prognostic Marker in Resected Pancreatic Ductal Adenocarcinoma Patients.

Simple SummaryOur explorative study used a microfluidic-based approach for circulating tumor cell (CTC) detection in 55 pancreatic ductal adenocarcinoma (PDAC) patients before treatment initiation (baseline) and during follow-up (FUP). For the first time, we assessed the expression of retinoic acid receptor responder 1 (RARRES1) on CTCs. CTCs were detected in 25.5% of patients at baseline, while the detection rate during FUP was higher (45.5%). Especially high CTC counts during FUP in resected patients were associated with early tumor relapse (p = 0.02). Combining CTC detection and RARRES1 protein expression showed that RARRES1-positive patients with high CTCs counts after curative operation during FUP had a worse prognosis (p = 0.001). In conclusion, RARRES1 is a new marker of interest for further research investigations on subtypes of CTCs in PDAC.Background: In pancreatic ductal adenocarcinoma (PDAC), the characterization of circulating tumor cells (CTCs) opens new insights into cancer metastasis as the leading cause of cancer-related death. Here, we focused on the expression of retinoic acid receptor responder 1 (RARRES1) on CTCs as a novel marker for treatment failure and early relapse. Methods: The stable isotope labeling of amino acids in cell culture (SILAC)—approach was applied for identifying and quantifying new biomarker proteins in PDAC cell lines HPDE and its chemoresistant counterpart, L3.6pl-Res. Fifty-five baseline and 36 follow-up (FUP) peripheral blood samples were processed via a marker-independent microfluidic-based CTC detection approach using RARRES1 as an additional marker. Results: SILAC-based proteomics identified RARRES1 as an abundantly expressed protein in more aggressive chemoresistant PDAC cells. At baseline, CTCs were detected in 25.5% of all PDAC patients, while FUP analysis (median: 11 months FUP) showed CTC detection in 45.5% of the resected patients. CTC positivity (≥3 CTC) at FUP was significantly associated with short recurrence-free survival (p = 0.002). Furthermore, detection of RARRES1 positive CTCs was indicative of an even earlier relapse after surgery (p = 0.001). Conclusions: CTC detection in resected PDAC patients during FUP is associated with a worse prognosis, and RARRES1 expression might identify an aggressive subtype of CTCs that deserves further investigation.

SILAC kinase screen identifies potential MASTL substrates

Microtubule-associated serine/threonine kinase-like (MASTL) has emerged as a critical regulator of mitosis and as a potential oncogene in a variety of cancer types. To date, Arpp-19/ENSA are the only known substrates of MASTL. However, with the roles of MASTL expanding and increased interest in development of MASTL inhibitors, it has become critical to determine if there are additional substrates and what the optimal consensus motif for MASTL is. Here we utilized a whole cell lysate in vitro kinase screen combined with stable isotope labelling of amino acids in cell culture (SILAC) to identify potential substrates and the residue preference of MASTL. Using the related AGC kinase family members AKT1/2, the kinase screen identified several known and new substrates highly enriched for the validated consensus motif of AKT. Applying this method to MASTL identified 59 phospho-sites on 67 proteins that increased in the presence of active MASTL. Subsequent in vitro kinase assays suggested that MASTL may phosphorylate hnRNPM, YB1 and TUBA1C under certain in vitro conditions. Taken together, these data suggest that MASTL may phosphorylate several additional substrates, providing insight into the ever-increasing biological functions and roles MASTL plays in driving cancer progression and therapy resistance.

Chemical Genetic Validation of CSNK2 Substrates Using an Inhibitor-Resistant Mutant in Combination with Triple SILAC Quantitative Phosphoproteomics.

Casein Kinase 2 (CSNK2) is an extremely pleiotropic, ubiquitously expressed protein kinase involved in the regulation of numerous key biological processes. Mapping the CSNK2-dependent phosphoproteome is necessary for better characterization of its fundamental role in cellular signalling. While ATP-competitive inhibitors have enabled the identification of many putative kinase substrates, compounds targeting the highly conserved ATP-binding pocket often exhibit off-target effects limiting their utility for definitive kinase-substrate assignment. To overcome this limitation, we devised a strategy combining chemical genetics and quantitative phosphoproteomics to identify and validate CSNK2 substrates. We engineered U2OS cells expressing exogenous wild type CSNK2A1 (WT) or a triple mutant (TM, V66A/H160D/I174A) with substitutions at residues important for inhibitor binding. These cells were treated with CX-4945, a clinical-stage inhibitor of CSNK2, and analyzed using large-scale triple SILAC (Stable Isotope Labelling of Amino Acids in Cell Culture) quantitative phosphoproteomics. In contrast to wild-type CSNK2A1, CSNK2A1-TM retained activity in the presence of CX-4945 enabling identification and validation of several CSNK2 substrates on the basis of their increased phosphorylation in cells expressing CSNK2A1-TM. Based on high conservation within the kinase family, we expect that this strategy can be broadly adapted for identification of other kinase-substrate relationships.

Vps68 cooperates with ESCRT-III in intraluminal vesicle formation.

The endosomal sorting complex required for transport (ESCRT)-III mediates budding and abscission of intraluminal vesicles (ILVs) into multivesicular endosomes. To further define the role of the yeast ESCRT-III-associated protein Mos10 (also known as Vps60) in ILV formation, we screened for new interaction partners by using stable isotope labeling of amino acids in cell culture (SILAC) and mass spectrometry. Here, we focused on the newly identified interaction partner Vps68. Our data suggest that Vps68 cooperates with ESCRT-III in ILV formation. The deletion of VPS68 caused a sorting defect similar to that of the SNF7 deletion strain when the cargo load was high. The composition of ESCRT-III was altered, the level of core components was higher and the level of associated proteins was lower in the VPS68 deletion strain. Our data further indicate that at some point in the functional cycle of ESCRT-III, Snf7 could be replaced by Mos10. Vps68 has an unusual membrane topology. Two of its potential membrane helices are amphipathic helices that localize to the luminal side of the endosomal membrane. Based on this membrane topology, we propose that Vps68 and ESCRT-III cooperate in the abscission step by weakening the luminal and cytosolic leaflets of the bilayer at the abscission site.

Quantitative Mass Spectrometry by SILAC in Haloferax volcanii.

The development of mass spectrometry (MS)-based proteomics methods has been critical in providing new insight about cellular processes and adaptations in all domains of life. While traditional MS-based methods are not inherently quantitative, technologies are now available to overcome this limitation. Of note, stable isotope labeling of amino acids in cell culture (SILAC) is reported as a reliable tool to label proteomes for quantitative MS-based proteomics that is accurate and flexible for multiplexing. The isotopically labeled lysine and arginine are easily incorporated into the proteome of cells auxotrophic for these amino acids. Microorganisms of the domain Archaea provide a fascinating alternative to understanding cellular adaptations and responses to environmental stresses. However, the availability of preferred SILAC-based quantitative analyses is limited. This protocol describes the use of SILAC to quantitatively analyze the proteome of Haloferax volcanii, a mesophilic halophilic archaeon that is easy to grow and requires no special equipment to maintain.

Quantitative proteomic screen identifies annexin A2 as a host target for Salmonella pathogenicity island-2 effectors SopD2 and PipB2

Intracellular pathogens need to establish an intracellular replicative niche to promote survival and replication within the hostile environment inside the host cell. Salmonella enterica serovar Typhimurium (S. Typhimurium) initiates formation of the unique Salmonella-containing vacuole and an extensive network of Salmonella-induced tubules in order to survive and thrive within host cells. At least six effectors secreted by the type III secretion system encoded within Salmonella pathogenicity island-2 (SPI-2), namely SifA, SopD2, PipB2, SteA, SseJ, and SseF, purportedly manipulate host cell intracellular trafficking and establish the intracellular replicative niche for S. Typhimurium. The phenotypes of these effectors are both subtle and complex, complicating elucidation of the mechanism underpinning host cell manipulation by S. Typhimurium. In this work we used stable isotope labeling of amino acids in cell culture (SILAC) and a S. Typhimurium mutant that secretes increased amounts of effectors to identify cognate effector binding partners during infection. Using this method, we identified the host protein annexin A2 (AnxA2) as a binding partner for both SopD2 and PipB2 and were able to confirm its binding to SopD2 and PipB2 by reciprocal pull down, although there was a low level of non-specific binding of SopD2-2HA and PipB2-2HA to the Ni-Sepharose beads present. We further showed that knockdown of AnxA2 altered the intracellular positioning of the Salmonella containing vacuole (SCV). This suggests that AnxA2 plays a role in the subcellular positioning of the SCV which could potentially be mediated through protein–protein interactions with either SopD2 or PipB2. This demonstrates the value of studying effector interactions using proteomic techniques and natural effector delivery during infection rather than transfection.

Identification and characterization of the mediator kinase-dependent myometrial stem cell phosphoproteome

To identify, in myometrial stem/progenitor cells, the presumptive cell of origin for uterine fibroids, substrates of Mediator-associated cyclin dependent kinase 8/19 (CDK8/19), which is known to be disrupted by uterine fibroid driver mutations in Mediator complex subunit 12 (MED12). Experimental study. Academic research laboratory. Women undergoing hysterectomy for uterine fibroids. Stable isotopic labeling of amino acids in cell culture (SILAC) coupled with chemical inhibition of CDK8/19 and downstream quantitative phosphoproteomics and transcriptomic analyses in myometrial stem/progenitor cells. High-confidence Mediator kinase substrates identified by SILAC-based quantitative phosphoproteomics were determined using an empirical Bayes analysis and validated orthogonally by invitro kinase assay featuring reconstituted Mediator kinase modules comprising wild-type or G44D mutant MED12 corresponding to the most frequent uterine fibroid driver mutation in MED12. Mediator kinase-regulated transcripts identified by RNA sequencing were linked to Mediator kinase substrates by computational analyses. A total of 296 unique phosphosites in 166 proteins were significantly decreased (≥ twofold) upon CDK8/19 inhibition, including 118 phosphosites in 71 nuclear proteins representing high-confidence Mediator kinase substrates linked to RNA polymerase II transcription, RNA processing and transport, chromatin modification, cytoskeletal architecture, and DNA replication and repair. Orthogonal validation confirmed a subset of these proteins, including Cut Like Homeobox 1 (CUX1) and Forkhead Box K1 (FOXK1), to be direct targets of MED12-dependent CDK8 phosphorylation in a manner abrogated by the most common uterine fibroid driver mutation (G44D) in MED12, implicating these substrates in disease pathogenesis. Transcriptome-wide profiling of Mediator kinase-inhibited myometrial stem/progenitor cells revealed alterations in cell cycle and myogenic gene expression programs to which Mediator kinase substrates could be linked directly. Among these, CUX1 is an established transcriptional regulator of the cell cycle whose corresponding gene on chromosome 7q is the locus for a recurrent breakpoint in uterine fibroids, linking MED12 and Mediator kinase with CUX1 for the first time in uterine fibroid pathogenesis. FOXK1, a transcriptional regulator of myogenic stem cell fate, was found to be coordinately enriched along with kinase, but not core, Mediator subunits in myometrial stem/progenitor cells compared with differentiated uterine smooth muscle cells. These studies identify a new catalog of pathologically and biologically relevant Mediator kinase substrates implicated in the pathogenesis of MED12 mutation-positive uterine fibroids, and further uncover a biochemical basis to link Mediator kinase activity with CUX1 and FOXK1 in the regulation of myometrial stem/progenitor cell fate.

Global analysis of HBV-mediated host proteome and ubiquitylome change in HepG2.2.15 human hepatoblastoma cell line

Hepatitis B virus (HBV) infection remains a major health issue worldwide and the leading cause of cirrhosis and hepatocellular carcinoma (HCC). It has been reported previously that HBV invasion can extensively alter transcriptome, the proteome of exosomes and host cell lipid rafts. The impact of HBV on host proteins through regulating their global post-translational modifications (PTMs), however, is not well studied. Viruses have been reported to exploit cellular processes by enhancing or inhibiting the ubiquitination of specific substrates. Nevertheless, host cell physiology in terms of global proteome and ubiquitylome has not been addressed yet. Here by using HBV-integrated HepG2.2.15 model cell line we first report that HBV significantly modify the host global ubiquitylome. As currently the most widely used HBV cell culture model, HepG2.2.15 can be cultivated for multiple generations for protein labeling, and can replicate HBV, express HBV proteins and secrete complete HBV Dane particles, which makes it a suitable cell line for ubiquitylome analysis to study HBV replication, hepatocyte immune response and HBV-related HCC progression. Our previous experimental results showed that the total ubiquitination level of HepG2.2.15 cell line was significantly higher than that of the corresponding parental HepG2 cell line. By performing a Ubiscan quantification analysis based on stable isotope labeling of amino acids in cell culture (SILAC) of HepG2.2.15 and HepG2 cell lines, we identified a total of 7188 proteins and the protein levels of nearly 19% of them were changed over 2-folds. We further identified 3798 ubiquitinated Lys sites in 1476 host proteins with altered ubiquitination in response to HBV. Our results also showed that the global proteome and ubiquitylome were negatively correlated, indicating that ubiquitination might be involved in the degradation of host proteins upon HBV integration. We first demonstrated the ubiquitination change of VAMP3, VAMP8, DNAJB6, RAB8A, LYN, VDAC2, OTULIN, SLC1A4, SLC1A5, HGS and TOLLIP. In addition, we described 5 novel host factors SLC1A4, SLC1A5, EIF4A1, TOLLIP and BRCC36 that efficiently reduced the amounts of secreted HBsAg and HBeAg. Overall, the HBV-mediated host proteome and ubiquitylome change we reported will provide a valuable resource for further investigation of HBV pathogenesis and host-virus interaction networks.

Discovery of Arginine Methylation, Phosphorylation, and Their Co-occurrence in Condensate-Associated Proteins in Saccharomyces cerevisiae.

The formation of condensates in membraneless organelles is thought to be driven by protein phase separation. Arginine methylation and serine/threonine phosphorylation are important in the phase separation process; however, these post-translational modifications are often present in intrinsically disordered regions that are difficult to analyze with standard proteomic techniques. To understand their presence and co-occurrence in condensate-associated proteins, here, we use a multiprotease and multi-tandem mass spectrometry (MS/MS) fragmentation approach, coupled with heavy methyl stable isotope labeling of amino acids in cell culture (SILAC) and phospho- or methyl-peptide enrichment. For Saccharomyces cerevisiae, we report a 50% increase in the known arginine methylproteome, involving 15 proteins that are all condensate-associated. Importantly, some of these proteins have arginine methylation on all predicted sites-providing evidence that this modification can be pervasive. We explored whether arginine-methylated, condensate-associated proteins are also phosphorylated and found 12 such proteins to carry phosphorylated serine or threonine. In Npl3, Ded1, and Sbp1, single peptides were found to carry both modifications, indicating a co-occurrence in close proximity and on the same protein molecule. These co-modifications occur in regions of disorder, whereas arginine methylation is typically on regions of disorder that are also basic. For phosphorylation, its association with charged regions of condensate-associated proteins was less consistent, although some regions with multisite phosphorylation sites were strongly acidic. We conclude that arginine-methylated proteins associated with condensates are typically also modified with protein phosphorylation.

Triple SILAC identified progestin-independent and dependent PRA and PRB interacting partners in breast cancer

Progesterone receptor (PR) isoforms, PRA and PRB, act in a progesterone-independent and dependent manner to differentially modulate the biology of breast cancer cells. Here we show that the differences in PRA and PRB structure facilitate the binding of common and distinct protein interacting partners affecting the downstream signaling events of each PR-isoform. Tet-inducible HA-tagged PRA or HA-tagged PRB constructs were expressed in T47DC42 (PR/ER negative) breast cancer cells. Affinity purification coupled with stable isotope labeling of amino acids in cell culture (SILAC) mass spectrometry technique was performed to comprehensively study PRA and PRB interacting partners in both unliganded and liganded conditions. To validate our findings, we applied both forward and reverse SILAC conditions to effectively minimize experimental errors. These datasets will be useful in investigating PRA- and PRB-specific molecular mechanisms and as a database for subsequent experiments to identify novel PRA and PRB interacting proteins that differentially mediated different biological functions in breast cancer.

Iron Deficiency Reprograms Phosphorylation Signaling and Reduces O-GlcNAc Pathways in Neuronal Cells.

Micronutrient sensing is critical for cellular growth and differentiation. Deficiencies in essential nutrients such as iron strongly affect neuronal cell development and may lead to defects in neuronal function that cannot be remedied by subsequent iron supplementation. To understand the adaptive intracellular responses to iron deficiency in neuronal cells, we developed and utilized a Stable Isotopic Labeling of Amino acids in Cell culture (SILAC)-based quantitative phosphoproteomics workflow. Our integrated approach was designed to comprehensively elucidate the changes in phosphorylation signaling under both acute and chronic iron-deficient cell models. In addition, we analyzed the differential cellular responses between iron deficiency and hypoxia (oxygen-deprived) in neuronal cells. Our analysis identified nearly 16,000 phosphorylation sites in HT-22 cells, a hippocampal-derived neuronal cell line, more than ten percent of which showed at least 2-fold changes in response to either hypoxia or acute/chronic iron deficiency. Bioinformatic analysis revealed that iron deficiency altered key metabolic and epigenetic pathways including the phosphorylation of proteins involved in iron sequestration, glutamate metabolism, and histone methylation. In particular, iron deficiency increased glutamine-fructose-6-phosphate transaminase (GFPT1) phosphorylation, which is a key enzyme in the glucosamine biosynthesis pathway and a target of 5′ AMP-activated protein kinase (AMPK), leading to reduced GFPT1 enzymatic activity and consequently lower global O-GlcNAc modification in neuronal cells. Taken together, our analysis of the phosphoproteome dynamics in response to iron and oxygen deprivation demonstrated an adaptive cellular response by mounting post-translational modifications that are critical for intracellular signaling and epigenetic programming in neuronal cells.

Targeting TSLP-Induced Tyrosine Kinase Signaling Pathways in CRLF2-Rearranged Ph-like ALL.

Philadelphia (Ph)-like acute lymphoblastic leukemia (ALL) is characterized by aberrant activation of signaling pathways and high risk of relapse. Approximately 50% of Ph-like ALL cases overexpress cytokine receptor-like factor 2 (CRLF2) associated with gene rearrangement. Activated by its ligand thymic stromal lymphopoietin (TSLP), CRLF2 signaling is critical for the development, proliferation, and survival of normal lymphocytes. To examine activation of tyrosine kinases regulated by TSLP/CRLF2, phosphotyrosine (P-Tyr) profiling coupled with stable isotope labeling of amino acids in cell culture (SILAC) was conducted using two CRLF2-rearranged (CRLF2r) Ph-like ALL cell lines stimulated with TSLP. As a result, increased P-Tyr was detected in previously reported TSLP-activated tyrosine kinases and substrates, including JAK1, JAK2, STAT5, and ERK1/2. Interestingly, TSLP also increased P-Tyr of insulin growth factor 1 receptor (IGF1R) and fibroblast growth factor receptor 1 (FGFR1), both of which can be targeted with small-molecule inhibitors. Fixed-ratio combination cytotoxicity assays using the tyrosine kinase inhibitors BMS-754807 and ponatinib that target IGF1R and FGFR1, respectively, revealed strong synergy against both cell line and patient-derived xenograft (PDX) models of CRLF2r Ph-like ALL. Further analyses also indicated off-target effects of ponatinib in the synergy, and novel association of the Ras-associated protein-1 (Rap1) signaling pathway with TSLP signaling in CRLF2r Ph-like ALL. When tested in vivo, the BMS-754807/ponatinib combination exerted minimal efficacy against 2 Ph-like ALL PDXs, associated with low achievable plasma drug concentrations. Although this study identified potential new targets in CRLF2r Ph-like ALL, it also highlights that in vivo validation of synergistic drug interactions is essential. IMPLICATION: Quantitative phosphotyrosine profiling identified potential therapeutic targets for high-risk CRLF2-rearranged Ph-like ALL.