Cell-specific Proteome Research Articles

Cell-specific cross-talk proteomics reveals cathepsin B signaling as a driver of glioblastoma malignancy near the subventricular zone.

Glioblastoma (GBM) is the most prevalent and aggressive malignant primary brain tumor. GBM proximal to the lateral ventricles (LVs) is more aggressive, potentially because of subventricular zone contact. Despite this, cross-talk between GBM and neural stem/progenitor cells (NSC/NPCs) is not well understood. Using cell-specific proteomics, we show that LV-proximal GBM prevents neuronal maturation of NSCs through induction of senescence. In addition, GBM brain tumor-initiating cells (BTICs) increase expression of cathepsin B (CTSB) upon interaction with NPCs. Lentiviral knockdown and recombinant protein experiments reveal that both cell-intrinsic and soluble CTSB promote malignancy-associated phenotypes in BTICs. Soluble CTSB stalls neuronal maturation in NPCs while promoting senescence, providing a link between LV-tumor proximity and neurogenesis disruption. Last, we show LV-proximal CTSB up-regulation in patients, showing the relevance of this cross-talk in human GBM biology. These results demonstrate the value of proteomic analysis in tumor microenvironment research and provide direction for new therapeutic strategies in GBM.

Abstract We049: Cell-Specific Metabolic Labelling Identifies hiPSC-CM Proteome In Vitro and In Vivo Post-Transplantation

Background: Human induced pluripotent stem cells (hiPSCs) and their derived cardiomyocytes (hiPSC-CMs) have been used for cell-based therapies for myocardial regeneration. Underlying paracrine signals derived from these hiPSC-CMs have been shown to play a critical role in improving the function of the ischemic myocardium. However, characterization of the transplanted cell proteome in vivo has been challenging due to the lack of tools to distinguish cell-specific proteome in a complex multicellular microenvironment. Methods: To establish cell-specific proteome labelling, we adopted the biorthogonal non-canonical amino acid tagging (BONCAT) system in hiPSCs. We expressed the mutant mouse tRNA synthetase gene, “L274GmMetRS” in hiPSCs (L274G-hiPSCs) using lentiviral transduction to enable incorporation of the non-canonical amino acid azidonorleucine (Anl) in these cells. We assessed the pluripotency and trilineage differentiation potential of the L274G-hiPSCs in vitro and in vivo (teratoma formation assay). Furthermore, we validated the cell-specific Anl incorporation in the L274G-hiPSC-CMs both in vitro (co-culture) and in vivo (post-transplantation). Results: Our studies showed that the L274G-hiPSCs could efficiently differentiate into all the three germ lineages and can be used to track the cell-specific proteome in their differentiated progenies, including L274G-hiPSC-CMs. Furthermore, immunostaining and western blots showed cell-specific BONCAT in L274G-hiPSC-CMs both in co-cultures (in vitro) and post-transplantation (in vivo). Conclusion: The newly-established L274G-hiPSC line can be used to track cell-specific proteome of hiPSC-CMs in vitro and in vivo, to characterize cell-specific proteomic responses and delineate mechanisms underlying cell therapies.

Bioorthogonal non-canonical amino acid tagging to track transplanted human induced pluripotent stem cell-specific proteome

BackgroundHuman induced pluripotent stem cells (hiPSCs) and their differentiated cell types have a great potential for tissue repair and regeneration. While the primary focus of using hiPSCs has historically been to regenerate damaged tissue, emerging studies have shown a more potent effect of hiPSC-derived paracrine factors on tissue regeneration. However, the precise contents of the transplanted hiPSC-derived cell secretome are ambiguous. This is mainly due to the lack of tools to distinguish cell-specific secretome from host-derived proteins in a complex tissue microenvironment in vivo.MethodsIn this study, we present the generation and characterization of a novel hiPSC line, L274G-hiPSC, expressing the murine mutant methionyl-tRNA synthetase, L274GMmMetRS, which can be used for tracking the cell specific proteome via biorthogonal non-canonical amino acid tagging (BONCAT). We assessed the trilineage differentiation potential of the L274G-hiPSCs in vitro and in vivo. Furthermore, we assessed the cell-specific proteome labelling in the L274G-hiPSC derived cardiomyocytes (L274G-hiPSC-CMs) in vitro following co-culture with wild type human umbilical vein derived endothelial cells and in vivo post transplantation in murine hearts.ResultsWe demonstrated that the L274G-hiPSCs exhibit typical hiPSC characteristics and that we can efficiently track the cell-specific proteome in their differentiated progenies belonging to the three germ lineages, including L274G-hiPSC-CMs. Finally, we demonstrated cell-specific BONCAT in transplanted L274G-hiPSC-CMs.ConclusionThe novel L274G-hiPSC line can be used to study the cell-specific proteome of hiPSCs in vitro and in vivo, to delineate mechanisms underlying hiPSC-based cell therapies for a variety of regenerative medicine applications.

MACSPI enables tissue-selective proteomic and interactomic analyses in multicellular organisms

Multicellular organisms are composed of many tissue types that have distinct morphologies and functions, which are largely driven by specialized proteomes and interactomes. To define the proteome and interactome of a specific type of tissue in an intact animal, we developed a localized proteomics approach called Methionine Analog-based Cell-Specific Proteomics and Interactomics (MACSPI). This method uses the tissue-specific expression of an engineered methionyl-tRNA synthetase to label proteins with a bifunctional amino acid 2-amino-5-diazirinylnonynoic acid in selected cells. We applied MACSPI in Caenorhabditis elegans, a model multicellular organism, to selectively label, capture, and profile the proteomes of the body wall muscle and the nervous system, which led to the identification of tissue-specific proteins. Using the photo-cross-linker, we successfully profiled HSP90 interactors in muscles and neurons and identified tissue-specific interactors and stress-related interactors. Our study demonstrates that MACSPI can be used to profile tissue-specific proteomes and interactomes in intact multicellular organisms.

The proteome landscape of the root cap reveals a role for the jacalin-associated lectin JAL10 in the salt-induced endoplasmic reticulum stress pathway

Rapid climate change has led to enhanced soil salinity, one of the major determinants of land degradation, resulting in low agricultural productivity. This has a strong negative impact on food security and environmental sustainability. Plants display various physiological, developmental, and cellular responses to deal with salt stress. Recent studies have highlighted the root cap as the primary stress sensor and revealed its crucial role in halotropism. The root cap covers the primary root meristem and is the first cell type to sense and respond to soil salinity, relaying the signal to neighboring cell types. However, it remains unclear how root-cap cells perceive salt stress and contribute to the salt-stress response. Here, we performed a root-cap cell-specific proteomics study to identify changes in the proteome caused by salt stress. The study revealed a very specific salt-stress response pattern in root-cap cells compared with non-root-cap cells and identified several novel proteins unique to the root cap. Root-cap-specific protein–protein interaction (PPI) networks derived by superimposing proteomics data onto known global PPI networks revealed that the endoplasmic reticulum (ER) stress pathway is specifically activated in root-cap cells upon salt stress. Importantly, we identified root-cap-specific jacalin-associated lectins (JALs) expressed in response to salt stress. A JAL10-GFP fusion protein was shown to be localized to the ER. Analysis of jal10 mutants indicated a role for JAL10 in regulating the ER stress pathway in response to salt. Taken together, our findings highlight the participation of specific root-cap proteins in salt-stress response pathways. Furthermore, root-cap-specific JAL proteins and their role in the salt-mediated ER stress pathway open a new avenue for exploring tolerance mechanisms and devising better strategies to increase plant salinity tolerance and enhance agricultural productivity.

CNSC-16. BIDIRECTIONAL SIGNALING BETWEEN GLIOBLASTOMA AND NEURAL PROGENITORS EXPLORED WITH CELL-SPECIFIC PROTEOMICS RESULTS IN INCREASED TUMOR MALIGNANCY AND ALTERED NEUROGENESIS IN THE SUBVENTRICULAR ZONE

Abstract Glioblastoma (GBM) is the most common and aggressive primary neoplasm of the central nervous system. The location of GBM contributes significantly to patient outcomes, where tumors contacting the lateral ventricles (LVs) have increased expression of stem cell genes, increased incidence of distal recurrence, and decreased overall survival. While the reasons for these findings are not fully understood, we hypothesize they arise due to interactions with the subventricular zone (SVZ), the largest neurogenic niche in mammals. We examined bidirectional signals between GBM cells and neural progenitor cells (NPCs) in vitro and in vivo using a combination of patient-derived intraoperative samples, preclinical animal models, and the methionine tRNA synthetase L274G (MetRS*) nascent proteomic labeling system, which allows for cell-specific proteomics analysis from complex in vivo systems. In co-culture with NPCs, GBM cells increase their viability, proliferation, migration, and expression of malignancy-promoting proteins, including migratory proteins such as TAGLN, PALLD, and STAT1. In vivo, tumor proximity to the SVZ results in increased proliferation, increased expression of stemness markers, decreased survival, and altered tumor-specific proteome. We then examined the reciprocal effect of GBM cells on NPC biology. In vitro and in vivo, GBM cells decreased NPC proliferation and induced decreased neuronal maturation of NPCs. To determine NPC proteomic changes in vivo, we created a transgenic mouse line Nestin-CreERT2; STOPflox R26-MetRS L274G, where upon tamoxifen administration Nestin+ NPCs express GFP and MetRS*. Our results indicate that LV-proximal GBM induces decreased expression of neuronal maturation proteins such as Eml1, VGLUT1, and EAAT2, and an increase in pro-migratory factors such as Lamc1 in NPCs. In conclusion, there are bidirectional proteomic interactions between GBM cells and NPCs that contributes to altered neurogenesis and increased tumor malignancy of LV-proximal GBM. The signaling factors identified in our studies will result in identification of novel therapeutic targets for GBM.

Single-cell transcriptomics and cell-specific proteomics reveals molecular signatures of sleep

Every day, we sleep for a third of the day. Sleep is important for cognition, brain waste clearance, metabolism, and immune responses. The molecular mechanisms governing sleep are largely unknown. Here, we used a combination of single-cell RNA sequencing and cell-type-specific proteomics to interrogate the molecular underpinnings of sleep. Different cell types in three important brain regions for sleep (brainstem, cortex, and hypothalamus) exhibited diverse transcriptional responses to sleep need. Sleep restriction modulates astrocyte-neuron crosstalk and sleep need enhances expression of specific sets of transcription factors in different brain regions. In cortex, we also interrogated the proteome of two major cell types: astrocytes and neurons. Sleep deprivation differentially alters the expression of proteins in astrocytes and neurons. Similarly, phosphoproteomics revealed large shifts in cell-type-specific protein phosphorylation. Our results indicate that sleep need regulates transcriptional, translational, and post-translational responses in a cell-specific manner.

Hierarchical Multi-Omics Data Integration and Modeling to Predict Novel Chemical-Induced Cell-Specific Proteomics Profiles for Systems Pharmacology

Hierarchical Multi-Omics Data Integration and Modeling to Predict Novel Chemical-Induced Cell-Specific Proteomics Profiles for Systems Pharmacology

Conditional Protein Rescue by Binding-Induced Protective Shielding.

Synthetic protein-level circuits offer an extra layer of cellular control on top of conventional gene-level circuits. Here, we describe a technology that allows conditional protein rescue (CPR) from proteasomal degradation using different protein inputs as masking agents. A target protein is fused to a degron tag and an affinity sensor domain. The use of nanobodies as the sensor domain offers a generalizable strategy to execute a wide range of protein-level circuits with ease. The utility of this new strategy was successfully demonstrated to distinguish cancer cells out of a healthy population using the HPV-specific E7 protein as a cellular marker. Because CPR can be programmed to execute more complex Boolean logic designs using cell-specific proteomes, this platform offers a highly modular and scalable framework for a wide range of applications based on synthetic protein circuits.

Cell-specific proteome analyses of human bone marrow reveal molecular features of age-dependent functional decline

Diminishing potential to replace damaged tissues is a hallmark for ageing of somatic stem cells, but the mechanisms remain elusive. Here, we present proteome-wide atlases of age-associated alterations in human haematopoietic stem and progenitor cells (HPCs) and five other cell populations that constitute the bone marrow niche. For each, the abundance of a large fraction of the ~12,000 proteins identified is assessed in 59 human subjects from different ages. As the HPCs become older, pathways in central carbon metabolism exhibit features reminiscent of the Warburg effect, where glycolytic intermediates are rerouted towards anabolism. Simultaneously, altered abundance of early regulators of HPC differentiation reveals a reduced functionality and a bias towards myeloid differentiation. Ageing causes alterations in the bone marrow niche too, and diminishes the functionality of the pathways involved in HPC homing. The data represent a valuable resource for further analyses, and for validation of knowledge gained from animal models.

Origin of exon skipping-rich transcriptomes in animals driven by evolution of gene architecture

BackgroundAlternative splicing, particularly through intron retention and exon skipping, is a major layer of pre-translational regulation in eukaryotes. While intron retention is believed to be the most prevalent mode across non-animal eukaryotes, animals have unusually high rates of exon skipping. However, when and how this high prevalence of exon skipping evolved is unknown. Since exon skipping can greatly expand proteomes, answering these questions sheds light on the evolution of higher organismal complexity in metazoans.ResultsWe used RNA-seq data to quantify exon skipping and intron retention frequencies across 65 eukaryotic species, with particular focus on early branching animals and unicellular holozoans. We found that only bilaterians have significantly increased their exon skipping frequencies compared to all other eukaryotic groups. Unlike in other eukaryotes, however, exon skipping in nearly all animals, including non-bilaterians, is strongly enriched for frame-preserving sequences, suggesting that exon skipping involvement in proteome expansion predated the increase in frequency. We also identified architectural features consistently associated with higher exon skipping rates within all studied eukaryotic genomes. Remarkably, these architectures became more prevalent during animal evolution, indicating co-evolution between genome architectures and exon skipping frequencies.ConclusionWe suggest that the increase of exon skipping rates in animals followed a two-step process. First, exon skipping in early animals became enriched for frame-preserving events. Second, bilaterian ancestors dramatically increased their exon skipping frequencies, likely driven by the interplay between a shift in their genome architectures towards more exon definition and recruitment of frame-preserving exon skipping events to functionally diversify their cell-specific proteomes.

Multiple Click-Selective tRNA Synthetases Expand Mammalian Cell-Specific Proteomics.

Bioorthogonal tools enable cell-type-specific proteomics, a prerequisite to understanding biological processes in multicellular organisms. Here we report two engineered aminoacyl-tRNA synthetases for mammalian bioorthogonal labeling: a tyrosyl ( ScTyrY43G) and a phenylalanyl ( MmPheT413G) tRNA synthetase that incorporate azide-bearing noncanonical amino acids specifically into the nascent proteomes of host cells. Azide-labeled proteins are chemoselectively tagged via azide-alkyne cycloadditions with fluorophores for imaging or affinity resins for mass spectrometric characterization. Both mutant synthetases label human, hamster, and mouse cell line proteins and selectively activate their azido-bearing amino acids over 10-fold above the canonical. ScTyrY43G and MmPheT413G label overlapping but distinct proteomes in human cell lines, with broader proteome coverage upon their coexpression. In mice, ScTyrY43G and MmPheT413G label the melanoma tumor proteome and plasma secretome. This work furnishes new tools for mammalian residue-specific bioorthogonal chemistry, and enables more robust and comprehensive cell-type-specific proteomics in live mammals.

HLA-G peptide preferences change in transformed cells: impact on the binding motif

HLA-G is known for its strictly restricted tissue distribution. HLA-G expression could be detected in immune privileged organs and many tumor entities such as leukemia, multiple myeloma, and non-Hodgkin and Hodgkin’s lymphoma. This functional variability from mediation of immune tolerance to facilitation of tumor immune evasion strategies might translate to a differential NK cell inhibition between immune-privileged organs and tumor cells. The biophysical invariability of the HLA-G heavy chain and its contrary diversity in immunity implicates a strong influence of the bound peptides on the pHLA-G structure. The aim was to determine if HLA-G displays a tissue-specific peptide repertoire. Therefore, using soluble sHLA-G technology, we analyzed the K562 and HDLM-2 peptide repertoires. Although both cell lines possess a comparable proteome and recruit HLA-G-restricted peptides through the same peptide-loading pathway, the peptide features appear to be cell specific. HDLM-2 derived HLA-G peptides are anchored by an Arg at p1 and K562-derived peptides are anchored by a Lys. At p2, no anchor motif could be determined while peptides were anchored at pΩ with a Leu and showed an auxiliary anchor motif Pro at p3. To appreciate if the peptide anchor alterations are due to a cell-specific differential peptidome, we performed analysis of peptide availability within the different cell types. Yet, the comparison of the cell-specific proteome and HLA-G-restricted ligandome clearly demonstrates a tissue-specific peptide selection by HLA-G molecules. This exclusive and unexpected observation suggests an exquisite immune function of HLA-G.

Labeling and identifying cell-specific proteomes in the mouse brain.

We develop an approach to tag proteomes synthesized by specific cell types in dissociated cortex, brain slices, and the brains of live mice. By viral-mediated expression of an orthogonal pyrrolysyl-tRNA synthetase-tRNAXXX pair in a cell type of interest and providing a non-canonical amino acid with a chemical handle, we selectively label neuronal or glial proteomes. The method enables the identification of proteins from spatially and genetically defined regions of the brain.

Tissue Specific Labeling in Proteomics.

Mass spectrometry-based proteomics is a powerful tool for identifying and quantifying proteins in biological samples. While it is routinely used for the characterization of simple cell line systems, the analysis of the cell specific proteome in multicellular organisms and tissues poses a significant challenge. Isolating a subset of cells from tissues requires mechanical and biochemical separation or sorting, a process which can alter cellular signaling, and thus, the composition of the proteome. Recently, several approaches for cell selective labeling of proteins, that include bioorthogonal amino acids, biotinylating enzymes, and genetic tools, have been developed. These tools facilitate the selective labeling of proteins, their interactome, or of specific cell types within a tissue or an organism, while avoiding the difficult and contamination-prone biochemical separation of cells from the tissue. In this review, we give an overview of existing techniques and their application in cell culture models and whole animals.

Cell-Specific Labeling for Analyzing Bidirectional Signaling by Mass Spectrometry.

Cell-specific proteome labeling enables global proteome-wide analysis of cell signaling in heterotypic co-cultures. Such approaches have provided unique insight in contact-initiated receptor tyrosine kinase signaling, transfer of proteomic material between heterotypic cells, and interactions between normal and oncogenic cells. Here we describe current methods for cell-specific labeling of heterotypic cells with isotopic labeled amino acids (e.g., SILAC and CTAP). We outline the advantages and disadvantages of individual approaches, describe typical experimental scenarios, and discuss where each experimental approach is optimally applied.

Abstract A34: Oncogenic KRAS regulates pancreatic cancer cell signaling via stromal reciprocation

Abstract Pancreatic Ductal Adenocarcinoma is characterized by a reactive stroma, which modifies tumor progression and response to therapy. Oncogenic mutations regulate signaling both within tumor cells and adjacent stromal cells. However, defining whether oncogenes can regulate tumor cell signaling and phenotypic behavior via stromal cells is of importance to understand mechanisms of disease progression and response to therapy. Here we show that oncogenic KRAS (KRASG12D) regulates pancreatic cancer cell signaling via stromal stellate cells. By combining cell-specific proteome labeling with multivariate phosphoproteomics, we analyzed heterocellular KRASG12D signaling in Pancreatic Ductal Adenocarcinoma (PDA) cells. Tumor cell KRASG12D engages heterotypic stellate cells, which subsequently instigate reciprocal signaling in the tumor cells. Reciprocal signaling employs additional kinases and doubles the number of regulated signaling nodes from cell-autonomous KRASG12D. Consequently, reciprocal KRASG12D produces a tumor cell phosphoproteome and total proteome that is distinct from cell-autonomous KRASG12D alone. Reciprocal signaling regulates tumor cell proliferation, apoptosis, and increases mitochondrial capacity via an IGF1R/AXL-AKT axis. These results demonstrate that oncogene signaling should be viewed as a heterocellular process and that our existing cell-autonomous perspective underrepresents the extent of oncogene signaling in cancer. Citation Format: Christopher Tape, Stephanie Ling, Maria Dimitriadi, Kelly McMahon, Jonathan Worboys, Hui S. Leong, Ida Norrie, Crispin Miller, George Poulogiannis, Douglas Lauffenburger, Claus Jorgensen.{Authors}. Oncogenic KRAS regulates pancreatic cancer cell signaling via stromal reciprocation. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A34.

Cell-specific proteomics with SORT-E

Cell-specific proteomics with SORT-E

Tagging and Enriching Proteins Enables Cell-Specific Proteomics

SummaryCell-specific proteomics in multicellular systems and whole animals is a promising approach to understand the differentiated functions of cells and tissues. Here, we extend our stochastic orthogonal recoding of translation (SORT) approach for the co-translational tagging of proteomes with a cyclopropene-containing amino acid in response to diverse codons in genetically targeted cells, and create a tetrazine-biotin probe containing a cleavable linker that offers a way to enrich and identify tagged proteins. We demonstrate that SORT with enrichment, SORT-E, efficiently recovers and enriches SORT tagged proteins and enables specific identification of enriched proteins via mass spectrometry, including low-abundance proteins. We show that tagging at distinct codons enriches overlapping, but distinct sets of proteins, suggesting that tagging at more than one codon enhances proteome coverage. Using SORT-E, we accomplish cell-specific proteomics in the fly. These results suggest that SORT-E will enable the definition of cell-specific proteomes in animals during development, disease progression, and learning and memory.

Oncogenic KRAS Regulates Tumor Cell Signaling via Stromal Reciprocation

SummaryOncogenic mutations regulate signaling within both tumor cells and adjacent stromal cells. Here, we show that oncogenic KRAS (KRASG12D) also regulates tumor cell signaling via stromal cells. By combining cell-specific proteome labeling with multivariate phosphoproteomics, we analyzed heterocellular KRASG12D signaling in pancreatic ductal adenocarcinoma (PDA) cells. Tumor cell KRASG12D engages heterotypic fibroblasts, which subsequently instigate reciprocal signaling in the tumor cells. Reciprocal signaling employs additional kinases and doubles the number of regulated signaling nodes from cell-autonomous KRASG12D. Consequently, reciprocal KRASG12D produces a tumor cell phosphoproteome and total proteome that is distinct from cell-autonomous KRASG12D alone. Reciprocal signaling regulates tumor cell proliferation and apoptosis and increases mitochondrial capacity via an IGF1R/AXL-AKT axis. These results demonstrate that oncogene signaling should be viewed as a heterocellular process and that our existing cell-autonomous perspective underrepresents the extent of oncogene signaling in cancer.Video