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BioRxiv : the Preprint Server For... Feb 2024During thymic development, most γδ T cells acquire innate-like characteristics that are critical for their function in tumor surveillance, infectious disease, and...
During thymic development, most γδ T cells acquire innate-like characteristics that are critical for their function in tumor surveillance, infectious disease, and tissue repair. The mechanisms, however, that regulate γδ T cell developmental programming remain unclear. Recently, we demonstrated that the SLAM-SAP signaling pathway regulates the development and function of multiple innate-like γδ T cell subsets. Here, we used a single-cell proteogenomics approach to identify SAP-dependent developmental checkpoints and to define the SAP-dependent γδ TCR repertoire. SAP deficiency resulted in both a significant loss of an immature γδT17 precursor population, and a significant increase in thymic γδ T cells. SAP-dependent diversion of embryonic day 17 thymic γδ T cell clonotypes into the αβ T cell developmental pathway was associated with a decreased frequency of mature clonotypes in neonatal thymus, and an altered γδ TCR repertoire in the periphery. Finally, we identify TRGV4/TRAV13-4(DV7)-expressing T cells as a novel, SAP-dependent Vγ4 γδT1 subset. Together, the data suggest that SAP-dependent γδ/αβ T cell lineage commitment regulates γδ T cell developmental programming and shapes the γδ TCR repertoire.
PubMed: 38260519
DOI: 10.1101/2024.01.10.575073 -
BioRxiv : the Preprint Server For... Jan 2024ZNRF3 and RNF43 are closely related transmembrane E3 ubiquitin ligases with significant roles in development and cancer. Conventionally, their biological functions have...
ZNRF3 and RNF43 are closely related transmembrane E3 ubiquitin ligases with significant roles in development and cancer. Conventionally, their biological functions have been associated with regulating WNT signaling receptor ubiquitination and degradation. However, our proteogenomic studies have revealed EGFR as the most negatively correlated protein with mRNA levels in multiple human cancers. Through biochemical investigations, we demonstrate that ZNRF3/RNF43 interact with EGFR via their extracellular domains, leading to EGFR ubiquitination and subsequent degradation facilitated by the E3 ligase RING domain. Overexpression of reduces EGFR levels and suppresses cancer cell growth and , whereas knockout of stimulates cell growth and tumorigenesis through upregulated EGFR signaling. Together, these data highlight ZNRF3 and RNF43 as novel E3 ubiquitin ligases of EGFR and establish the inactivation of ZNRF3/RNF43 as a driver of increased EGFR signaling, ultimately promoting cancer progression. This discovery establishes a connection between two fundamental signaling pathways, EGFR and WNT, at the level of cytoplasmic membrane receptor, uncovering a novel mechanism underlying the frequent co-activation of EGFR and WNT signaling in development and cancer.
PubMed: 38260423
DOI: 10.1101/2024.01.10.574969 -
International Journal of Molecular... Jan 2024SpliceProt 2.0 is a public proteogenomics database that aims to list the sequence of known proteins and potential new proteoforms in human, mouse, and rat proteomes....
SpliceProt 2.0 is a public proteogenomics database that aims to list the sequence of known proteins and potential new proteoforms in human, mouse, and rat proteomes. This updated repository provides an even broader range of computationally translated proteins and serves, for example, to aid with proteomic validation of splice variants absent from the reference UniProtKB/SwissProt database. We demonstrate the value of SpliceProt 2.0 to predict orthologous proteins between humans and murines based on transcript reconstruction, sequence annotation and detection at the transcriptome and proteome levels. In this release, the annotation data used in the reconstruction of transcripts based on the methodology of ternary matrices were acquired from new databases such as Ensembl, UniProt, and APPRIS. Another innovation implemented in the pipeline is the exclusion of transcripts predicted to be susceptible to degradation through the NMD pathway. Taken together, our repository and its applications represent a valuable resource for the proteogenomics community.
Topics: Rats; Mice; Humans; Animals; Proteomics; Databases, Protein; Knowledge Bases; Proteogenomics; Proteome
PubMed: 38256255
DOI: 10.3390/ijms25021183 -
Clinical Proteomics Jan 2024Although uterine serous carcinoma (USC) represents a small proportion of all uterine cancer cases, patients with this aggressive subtype typically have high rates of...
BACKGROUND
Although uterine serous carcinoma (USC) represents a small proportion of all uterine cancer cases, patients with this aggressive subtype typically have high rates of chemotherapy resistance and disease recurrence that collectively result in a disproportionately high death rate. The goal of this study was to provide a deeper view of the tumor microenvironment of this poorly characterized uterine cancer variant through multi-region microsampling and quantitative proteomics.
METHODS
Tumor epithelium, tumor-involved stroma, and whole "bulk" tissue were harvested by laser microdissection (LMD) from spatially resolved levels from nine USC patient tumor specimens and underwent proteomic analysis by mass spectrometry and reverse phase protein arrays, as well as transcriptomic analysis by RNA-sequencing for one patient's tumor.
RESULTS
LMD enriched cell subpopulations demonstrated varying degrees of relatedness, indicating substantial intratumor heterogeneity emphasizing the necessity for enrichment of cellular subpopulations prior to molecular analysis. Known prognostic biomarkers were quantified with stable levels in both LMD enriched tumor and stroma, which were shown to be highly variable in bulk tissue. These USC data were further used in a comparative analysis with a data generated from another serous gynecologic malignancy, high grade serous ovarian carcinoma, and have been added to our publicly available data analysis tool, the Heterogeneity Analysis Portal ( https://lmdomics.org/ ).
CONCLUSIONS
Here we identified extensive three-dimensional heterogeneity within the USC tumor microenvironment, with disease-relevant biomarkers present in both the tumor and the stroma. These data underscore the critical need for upfront enrichment of cellular subpopulations from tissue specimens for spatial proteogenomic analysis.
PubMed: 38254014
DOI: 10.1186/s12014-024-09451-2 -
Molecular & Cellular Proteomics : MCP Feb 2024Although the human gene annotation has been continuously improved over the past 2 decades, numerous studies demonstrated the existence of a "dark proteome", consisting...
Although the human gene annotation has been continuously improved over the past 2 decades, numerous studies demonstrated the existence of a "dark proteome", consisting of proteins that were critical for biological processes but not included in widely used gene catalogs. The Genotype-Tissue Expression project generated more than 15,000 RNA-seq datasets from multiple tissues, which modeled 30 million transcripts in the human genome. To provide a resource of high-confidence novel proteins from the dark proteome, we screened 50,000 mass spectrometry runs from over 900 projects to identify proteins translated from the Genotype-Tissue Expression transcript model with proteomic support. We also integrated 3.8 million common genetic variants from the gnomAD database to improve peptide identification. As a result, we identified 170,529 novel peptides with proteomic evidence, of which 6048 passed the strictest standard we defined and were supported by PepQuery. We provided a user-friendly website (https://ncorf.genes.fun/) for researchers to check the evidence of novel peptides from their studies. The findings will improve our understanding of coding genes and facilitate genomic data interpretation in biomedical research.
Topics: Humans; Proteogenomics; Proteome; Proteomics; Peptides; Genome, Human
PubMed: 38242438
DOI: 10.1016/j.mcpro.2024.100719 -
Respiratory Research Jan 2024A decline in forced expiratory volume (FEV1) is a hallmark of respiratory diseases that are an important cause of morbidity among the elderly. While some data exist on...
BACKGROUND
A decline in forced expiratory volume (FEV1) is a hallmark of respiratory diseases that are an important cause of morbidity among the elderly. While some data exist on biomarkers that are related to FEV1, we sought to do a systematic analysis of causal relations of biomarkers with FEV1.
METHODS
Data from the population-based AGES-Reykjavik study were used. Serum proteomic measurements were done using 4782 DNA aptamers (SOMAmers). Data from 1479 participants with spirometric data were used to assess the association of SOMAmer measurements with FEV1 using linear regression. Bi-directional two-sample Mendelian randomisation (MR) analyses were done to assess causal relations of observationally associated SOMAmers with FEV1, using genotype and SOMAmer data from 5368 AGES-Reykjavik participants and genetic associations with FEV1 from a publicly available GWAS (n = 400,102).
RESULTS
In observational analyses, 530 SOMAmers were associated with FEV1 after multiple testing adjustment (FDR < 0.05). The most significant were Retinoic Acid Receptor Responder 2 (RARRES2), R-Spondin 4 (RSPO4) and Alkaline Phosphatase, Placental Like 2 (ALPPL2). Of the 257 SOMAmers with genetic instruments available, eight were associated with FEV1 in MR analyses. Three were directionally consistent with the observational estimate, Thrombospondin 2 (THBS2), Endoplasmic Reticulum Oxidoreductase 1 Beta (ERO1B) and Apolipoprotein M (APOM). THBS2 was further supported by a colocalization analysis. Analyses in the reverse direction, testing whether changes in SOMAmer levels were caused by changes in FEV1, were performed but no significant associations were found after multiple testing adjustments.
CONCLUSIONS
In summary, this large scale proteogenomic analyses of FEV1 reveals circulating protein markers of FEV1, as well as several proteins with potential causality to lung function.
Topics: Humans; Female; Pregnancy; Aged; Forced Expiratory Volume; Lung; Proteomics; Placenta; Biomarkers
PubMed: 38238732
DOI: 10.1186/s12931-023-02587-z -
Cell Reports. Medicine Jan 2024Acute myeloid leukemia is a poor-prognosis cancer commonly stratified by genetic aberrations, but these mutations are often heterogeneous and fail to consistently...
Acute myeloid leukemia is a poor-prognosis cancer commonly stratified by genetic aberrations, but these mutations are often heterogeneous and fail to consistently predict therapeutic response. Here, we combine transcriptomic, proteomic, and phosphoproteomic datasets with ex vivo drug sensitivity data to help understand the underlying pathophysiology of AML beyond mutations. We measure the proteome and phosphoproteome of 210 patients and combine them with genomic and transcriptomic measurements to identify four proteogenomic subtypes that complement existing genetic subtypes. We build a predictor to classify samples into subtypes and map them to a "landscape" that identifies specific drug response patterns. We then build a drug response prediction model to identify drugs that target distinct subtypes and validate our findings on cell lines representing various stages of quizartinib resistance. Our results show how multiomics data together with drug sensitivity data can inform therapy stratification and drug combinations in AML.
Topics: Humans; Proteomics; Proteogenomics; Leukemia, Myeloid, Acute; Genomics; Mutation
PubMed: 38232702
DOI: 10.1016/j.xcrm.2023.101359 -
Cell Reports. Medicine Jan 2024Lung adenocarcinoma follows a stepwise progression from pre-invasive to invasive. However, there remains a knowledge gap regarding molecular events from pre-invasive to...
Lung adenocarcinoma follows a stepwise progression from pre-invasive to invasive. However, there remains a knowledge gap regarding molecular events from pre-invasive to invasive. Here, we conduct a comprehensive proteogenomic analysis comprising whole-exon sequencing, RNA sequencing, and proteomic and phosphoproteomic profiling on 98 pre-invasive and 99 invasive lung adenocarcinomas. The deletion of chr4q12 contributes to the progression from pre-invasive to invasive adenocarcinoma by downregulating SPATA18, thus suppressing mitophagy and promoting cell invasion. Proteomics reveals diverse enriched pathways in normal lung tissues and pre-invasive and invasive adenocarcinoma. Proteomic analyses identify three proteomic subtypes, which represent different stages of tumor progression. We also illustrate the molecular characterization of four immune clusters, including endothelial cells, B cells, DCs, and immune depression subtype. In conclusion, this comprehensive proteogenomic study characterizes the molecular architecture and hallmarks from pre-invasive to invasive lung adenocarcinoma, guiding the way to a deeper understanding of the tumorigenesis and progression of this disease.
Topics: Humans; Proteogenomics; Lung Neoplasms; Proteomics; Endothelial Cells; Adenocarcinoma of Lung; Adenocarcinoma
PubMed: 38183982
DOI: 10.1016/j.xcrm.2023.101358 -
Scientific Data Jan 2024A wealth of proteogenomic data has been generated using cancer samples to deepen our understanding of the mechanisms of cancer and how biological networks are altered in...
A wealth of proteogenomic data has been generated using cancer samples to deepen our understanding of the mechanisms of cancer and how biological networks are altered in association with somatic mutation of tumor suppressor genes, such as TP53 and PTEN. To generate functional signatures of TP53 or PTEN loss, we profiled the RNA and phosphoproteomes of the MCF10A epithelial cell line, along with its congenic TP53- or PTEN-knockout derivatives, upon perturbation with the monofunctional DNA alkylating agent methyl methanesulfonate (MMS) vs. mock treatment. To enable quantitative and reproducible mass spectrometry data generation, the cell lines were SILAC-labeled (stable isotope labeling with amino acids in cell culture), and the experimental design included label swapping and biological replicates. All data are publicly available and may be used to advance our understanding of the TP53 and PTEN tumor suppressor genes and to provide functional signatures for bioinformatic analyses of proteogenomic datasets.
Topics: Humans; DNA Damage; Epithelial Cells; Mutation; Neoplasms; PTEN Phosphohydrolase; RNA; Tumor Suppressor Protein p53
PubMed: 38177134
DOI: 10.1038/s41597-023-02829-1 -
Molecular Neurodegeneration Jan 2024Triggering receptor expressed on myeloid cells 2 (TREM2) plays a critical role in microglial activation, survival, and apoptosis, as well as in Alzheimer's disease (AD)...
Triggering receptor expressed on myeloid cells 2 (TREM2) plays a critical role in microglial activation, survival, and apoptosis, as well as in Alzheimer's disease (AD) pathogenesis. We previously reported the MS4A locus as a key modulator for soluble TREM2 (sTREM2) in cerebrospinal fluid (CSF). To identify additional novel genetic modifiers of sTREM2, we performed the largest genome-wide association study (GWAS) and identified four loci for CSF sTREM2 in 3,350 individuals of European ancestry. Through multi-ethnic fine mapping, we identified two independent missense variants (p.M178V in MS4A4A and p.A112T in MS4A6A) that drive the association in MS4A locus and showed an epistatic effect for sTREM2 levels and AD risk. The novel TREM2 locus on chr 6 contains two rare missense variants (rs75932628 p.R47H, P=7.16×10; rs142232675 p.D87N, P=2.71×10) associated with sTREM2 and AD risk. The third novel locus in the TGFBR2 and RBMS3 gene region (rs73823326, P=3.86×10) included a regulatory variant with a microglia-specific chromatin loop for the promoter of TGFBR2. Using cell-based assays we demonstrate that overexpression and knock-down of TGFBR2, but not RBMS3, leads to significant changes of sTREM2. The last novel locus is located on the APOE region (rs11666329, P=2.52×10), but we demonstrated that this signal was independent of APOE genotype. This signal colocalized with cis-eQTL of NECTIN2 in the brain cortex and cis-pQTL of NECTIN2 in CSF. Overexpression of NECTIN2 led to an increase of sTREM2 supporting the genetic findings. To our knowledge, this is the largest study to date aimed at identifying genetic modifiers of CSF sTREM2. This study provided novel insights into the MS4A and TREM2 loci, two well-known AD risk genes, and identified TGFBR2 and NECTIN2 as additional modulators involved in TREM2 biology.
Topics: Humans; Alzheimer Disease; Receptor, Transforming Growth Factor-beta Type II; Genome-Wide Association Study; Microglia; Apolipoproteins E; Biomarkers; Membrane Glycoproteins; Receptors, Immunologic
PubMed: 38172904
DOI: 10.1186/s13024-023-00687-4