Biology subjects

Uhlen, M.

Publications and source records attributed to Uhlen, M..

50 records · Page 3Linked to original sources

bioRxiv2020

Mapping of the nucleolar proteome reveals spatiotemporal organization related to intrinsic protein disorder

The nucleolus is essential for ribosome biogenesis and is involved in many other cellular functions. We performed a systematic spatiotemporal dissection of the human nucleolar proteome using confocal microscopy. In total, 1,318 nucleolar proteins were identified; 287 were localized to fibrillar components, and 157 were enriched along the nucleoplasmic border, indicating a potential fourth nucleolar subcompartment (nucleoli rim). We found 65 nucleolar proteins (36 uncharacterized) to relocate to the chromosomal periphery during mitosis. Interestingly, we observed temporal partitioning into two recruitment phenotypes: early (prometaphase) and late (after metaphase), suggesting phase-specific functions. We further show that expression of MKI67 is critical for this temporal partitioning. We provide the first proteome-wide analysis of intrinsic protein disorder for the human nucleolus and show that nucleolar proteins in general, and mitotic chromosome proteins in particular, have significantly higher intrinsic disorder level compared to cytosolic proteins. In summary, this study provides a comprehensive and essential resource of spatiotemporal expression data for the nucleolar proteome as part of the Human Protein Atlas.

cell biology↗

bioRxiv2020

Human immune system variation during one year

The human immune system varies extensively between individuals, but variation within individuals over time has not been well characterized. Systems-level analyses allow for simultaneous quantification of many interacting immune system components, and the inference of global regulatory principles. Here we present a longitudinal, systems-level analysis in 99 healthy adults, 50 to 65 years of age and sampled every 3rd month during one year. We describe the structure of inter-individual variation and characterize extreme phenotypes along a principal curve. From coordinated measurement fluctuations, we infer relationships between 115 immune cell populations and 750 plasma proteins constituting the blood immune system. While most individuals have stable immune systems, the degree of longitudinal variability is an individual feature. The most variable individuals, in the absence of overt infections, exhibited markers of poor metabolic health suggestive of a functional link between metabolic and immunologic homeostatic regulation. HIGHLIGHTSLongitudinal variation in immune cell composition during one year Inter-individual variation can be described along a principal curve Immune cell and protein relationships are inferred Variability over time is an individual feature correlating with markers of poor metabolic health

immunology↗

bioRxiv2019

Classification of clear cell renal cell carcinoma based on PKM alternative splicing

Clear cell renal cell carcinoma (ccRCC) accounts for 70-80% of kidney cancer diagnoses and displays high molecular and histologic heterogeneity. Hence, it is necessary to reveal the underlying molecular mechanisms involved in progression of ccRCC to better stratify the patients and design effective treatment strategies. Here, we analyzed the survival outcome of ccRCC patients as a consequence of the differential expression of four transcript isoforms of the pyruvate kinase muscle type (PKM). We first extracted a classification biomarker consisting of eight gene pairs whose within-sample relative expression orderings (REOs) could be used to robustly classify the patients into two groups with distinct molecular characteristics and survival outcomes. Next, we validated our findings in a validation cohort and an independent Japanese ccRCC cohort. We finally performed drug repositioning analysis based on transcriptomic expression profiles of drug-perturbed cancer cell lines and proposed that paracetamol, nizatidine, dimethadione and conessine can be repurposed to treat the patients in one of the subtype of ccRCC whereas chenodeoxycholic acid, fenoterol and hexylcaine can be repurposed to treat the patients in the other subtype.

systems biology↗

bioRxiv2019

MOBN: an interactive database of multi-omics biological networks

It is essential to reveal the associations between different omics data for a comprehensive understanding of the altered biological process in human wellness and disease. To date, very few studies have focused on collecting and exhibiting multi-omics associations in a single database. Here, we present iNetModels, an interactive database and visualization platform of Multi-Omics Biological Networks (MOBNs). This platform describes the associations between the clinical chemistry, anthropometric parameters, plasma proteomics and metabolomics as well as metagenomics for oral and gut microbiome obtained from the same individuals. Moreover, iNetModels includes tissue- and cancer-specific Gene Co-expression Networks (GCNs) for exploring the connections between the specific genes. This platform allows the user to interactively explore a single feature's association with other omics data and customize its particular context (e.g. male/female specific). The users can also register their own data for sharing and visualization of the MOBNs and GCNs. Moreover, iNetModels allows users who do not have a bioinformatics background to facilitate human wellness and diseases research. iNetModels can be accessed freely at https://inetmodels.com without any limitation.

bioinformatics↗

bioRxiv2019

RMetD2: a tool for integration of relative transcriptomics data into Genome-scale metabolic models

Relative Metabolic Differences version 2 (RMetD2) is a tool for integration of differentially expressed (DE) genes into genome-scale metabolic models (GEMs) for revealing the altered metabolism between two biological conditions. This method provides a robust evaluation of the metabolism by using flux ranges instead of a single set of flux distributions. RMetD2 classifies reactions into three different groups, namely up-regulated, down-regulated and unchanged, which enables systematic interpretation of the metabolic differences between two different conditions. We employed this method in three different case studies using mice and human datasets, and compared it with state-of-the-art methods used for studying condition-specific metabolic differences using GEMs. We observed that RMetD2 is capable of capturing experimentally-observed features that are missed by other methods, highlighting its potential use in biotechnology and systems medicine applications. RMetD2 is implemented in Matlab and it is available without any limitation at https://sourceforge.net/projects/rmetd.

bioinformatics↗

bioRxiv2019

Discovery of functional alternatively spliced PKM transcripts in human cancers

The association of pyruvate kinase muscle type (PKM) with survival of cancer patients is controversial. Here, we focus on different transcripts of PKM and investigate the association between their mRNA expression and the clinical survival of the patients in 25 different cancers. We find that the transcript encoding PKM2, and three other functional transcripts are prognostic in multiple cancers. Our integrative analysis shows that the functions of these four transcripts are highly conservative in different cancers. Next, we validate the prognostic effect of these transcripts in an independent kidney renal clear-cell carcinoma (KIRC) cohort and identify a prognostic signature which could distinguish high- and low-risk KIRC patients. Finally, we reveal the functional role of alternatively spliced PKM transcripts in KIRC, and discover the protein products of different transcripts of PKM. Our analysis demonstrated that alternatively spliced transcripts of not only PKM but also other genes should be considered in cancer studies, since it may enable the discovery and targeting of the right protein product for development of the efficient treatment strategies.

systems biology↗

bioRxiv2019

Multiplexed Analysis of the Secretin-like GPCR-RAMP Interactome

Although receptor activity-modifying proteins (RAMPs) have been shown to modulate the functions of several different G protein-coupled receptors (GPCRs), potential direct interactions among the three known RAMPs and hundreds of GPCRs has never been investigated. We engineered three epitope-tagged RAMPs and 23 epitope-tagged GPCRs, focusing on the secretin-like family of GPCRs, and developed a suspension bead array (SBA) immunoassay designed to detect RAMP-GPCR complexes. We then used 64 antibodies raised against native RAMPs and GPCRs, along with four antibodies targeting the epitope tags, to multiplex the SBA assay to detect and measure all possible combinations of interaction among the 23 GPCRs and three RAMPs. The results of the SBA assay provide a complete interactome of secretin-like GPCRs with RAMPs. We demonstrate direct interaction of previously reported secretin-like GPCRs whose functions are modulated by RAMPs. We also discovered novel sets of GPCR-RAMP interacting pairs, and found additional secretin-like GPCRs, chemokine receptors and orphan receptors that interact with RAMPs. Using in situ roximity ligation assay, we verified a subset of these novel GPCR-RAMP interactions in cell membranes. In total, we found GPCR-RAMP interactions for the majority of the 23 GPCRs tested. Each GPCR interacted with either all three RAMPs or with RAMP2 and RAMP3, with the exception of one GPCR that interacted with just RAMP3. In summary, we describe an SBA strategy that will be useful to search for GPCR-RAMP interactions in cell lines and tissues, and conclude that GPCR-RAMP interactions are more common than previously appreciated.

biochemistry↗

bioRxiv2019

A systems-based map of human brain cell-type enriched genes and malignancy-associated endothelial changes

Changes in the endothelium of the cerebral vasculature can contribute to inflammatory, thrombotic and malignant disorders. The importance of defining cell type-specific genes and how they are modified in disease is increasingly recognised. Here, we developed a bioinformatics-based approach to identify normal brain cell-enriched genes, using bulk RNAseq data from 238 normal human cortex samples from 2 independent cohorts. We compared endothelial cell-enriched gene profiles with astrocyte, oligodendrocyte and neuron profiles. Global modifications to the endothelium in malignant disease were characterised, using RNAseq data from 516 human lower grade gliomas and 401 human glioblastoma multiforme samples. Lower grade glioma appeared to be an endothelial intermediate between normal brain and glioblastoma multiforme. We identify the most highly glioblastoma multiforme-specific endothelial cell biomarkers, providing potential diagnostic or therapeutic targets. In summary, we provide a roadmap of endothelial cell identity in normal and malignant brain tissue, using a bioinformatics-based method developed to resolve bulk RNAseq datasets into constituent cell type-enriched profiles.

cell biology↗

bioRxiv2019

Systematic Development Of Sandwich Immunoassays For The Plasma Secretome

The plasma proteome offers a clinically useful window into human health and disease. With recent progress made on the development of highly multiplexed immunoassays with high sample throughput, a remaining need is to establish a pipeline for validating the individual proteins that build such bio-signatures by using targeted assays. In order to streamline such efforts, we developed a workflow to build dual binder sandwich immunoassays (SIA) and chose to evaluate this on proteins predicted to be secreted form cells and tissues. Utilizing the multiplexing capacities of the bead array technology, we first screened ~ 1,800 unique antibody pairs against 209 protein targets and collected data from dilution series of recombinant proteins as well as EDTA plasma. Employing 624 unique antibodies from the Human Protein Atlas, we obtained dilution-dependent curves in plasma and concentration-dependent curves of full-length proteins for 102 (49%) of the targets. For 22 protein assays, the longitudinal, inter-individual and technical performance was determined in a set of plasma samples collected from 18 healthy subjects every third month over one year. Lastly, we compared 14 of these assays with SIAs composed of other binders, proximity extension assays and affinity-free targeted mass spectrometry. Our workflow provides a multiplexed approach to screen for SIA pairs that suggests using at least three antibodies per target. This design is applicable for a wider range of targets of the plasma proteome, while the assays can be applied for discovery but also to validate emerging candidates derived from other platforms.

biochemistry↗

bioRxiv2018

The human secretome - the proteins secreted from human cells

The proteins secreted by human tissues (the secretome) are important for the basic understanding of human biology, but also for identification of potential targets for future diagnosis and therapy. Here, we present an annotation of all predicted secreted proteins (n=2,623) with information about their spatial distribution in the human body. A high-throughput mammalian cell factory was established to create a resource of recombinant full-length proteins. This resource was used for phenotypic assays involving {beta}-cell dedifferentiation and for development of targeted proteomics assays. A comparison between host cells, including omics analysis, shows that many of the proteins that failed to be generated in CHO cells could be rescued in human HEK293 cells. In conclusion, the human secretome has been mapped and characterized to facilitate further exploration of the human secretome.

molecular biology↗

bioRxiv2018

Screening a Resource of Recombinant Protein Fragments for Targeted Proteomics

The availability of proteomics resources hosting protein and peptide standards, as well as the data describing their analytical performances, will continue to enhance our current capabilities to develop targeted proteomics methods for quantitative biology. This study describes the analysis of a resource of 26,840 individually purified recombinant protein fragments corresponding to more than 16,000 human protein-coding genes. The resource was screened to identify proteotypic peptides suitable for targeted proteomics efforts and we report LC-MS/MS assay coordinates for more than 25,000 proteotypic peptides, corresponding to more than 10,000 unique proteins. Additionally, peptide formation and digestion kinetics were, for a subset of the standards, monitored using a time-course protocol involving parallel digestion of isotope-labelled recombinant protein standards and endogenous human plasma proteins. We show that the strategy by adding isotope-labelled recombinant proteins prior to trypsin digestion enables short digestion protocols ([≤]60 min) with robust quantitative precision. In a proof-of-concept study, we quantified 23 proteins in human plasma using assay parameters defined in our study and used the standards to describe distinct clusters of individuals linked to different levels of LPA, APOE, SERPINA5 and TFRC. In summary, we describe the use and utility of a resource of recombinant proteins to identify proteotypic peptides useful for targeted proteomics assay development.

biochemistry↗

bioRxiv2018

Levels of histidine-rich glycoprotein in human blood predict accelerated aging

1.Despite recognizing aging as risk factor of human diseases, little is still known about the molecular traits of biological age and mortality risk. To identify age-associated proteins circulating human blood, we screened 156 subjects aged 50-92 years using an exploratory and multiplexed affinity proteomics approach. We corroborated the top age-associated protein profile (adjusted P < 0.001) in eight additional study sets (N = 4,044 individuals), and confirmed a consistent age-associated increase (P = 6.61 x 10-6) by meta-analysis. Applying antibody validation determined circulating histidine-rich glycoprotein (HRG) as the target, and we observed that sequence variants influenced the antibodies ability to bind to the protein. Profiles of circulating HRG were associated to several clinical traits and predicted the risk of mortality during a follow-up period of 8.5 years (IQR = 7.7-9.3 years) after blood sampling (HR = 1.25 per SD; 95% CI = 1.12-1.39; P = 7.41 x 10-5). In conclusion, our affinity proteomics analysis found associations between the molecular traits of circulating HRG with age and all-cause mortality. This suggests that the profiles of multi-purpose protein HRG could serve as an accessible indicator of physiological processes related to aging.

epidemiology↗

bioRxiv2018

Quantification and discovery of sequence determinants of protein per mRNA amount in 29 human tissues

Despite their importance in determining protein abundance, a comprehensive catalogue of sequence features controlling protein-to-mRNA (PTR) ratios and a quantification of their effects is still lacking. Here we quantified PTR ratios for 11,575 proteins across 29 human tissues using matched transcriptomes and proteomes. We analyzed the contribution of known sequence determinants of protein synthesis and degradation and 15 novel mRNA and protein sequence motifs that we found by association testing. While the dynamic range of PTR ratios spans more than 2 orders of magnitude, our integrative model predicts PTR ratios at a median precision of 3.2-fold. A reporter assay provided significant functional support for two novel UTR motifs and a proteome-wide competition-binding assay identified motif-specific bound proteins for one motif. Moreover, our direct comparison of protein to RNA levels led to a new metrics of codon optimality. Altogether, this study shows that a large fraction of PTR ratio variance across genes can be predicted from sequence and identified many new candidate post-transcriptional regulatory elements in the human genome.

systems biology↗

bioRxiv2018

A deep proteome and transcriptome abundance atlas of 29 healthy human tissues

Genome-, transcriptome- and proteome-wide measurements provide valuable insights into how biological systems are regulated. However, even fundamental aspects relating to which human proteins exist, where they are expressed and in which quantities are not fully understood. Therefore, we have generated a systematic, quantitative and deep proteome and transcriptome abundance atlas from 29 paired healthy human tissues from the Human Protein Atlas Project and representing human genes by 17,615 transcripts and 13,664 proteins. The analysis revealed that few proteins show truly tissue-specific expression, that vast differences between mRNA and protein quantities within and across tissues exist and that the expression levels of proteins are often more stable across tissues than those of transcripts. In addition, only ~2% of all exome and ~7% of all mRNA variants could be confidently detected at the protein level showing that proteogenomics remains challenging, requires rigorous validation using synthetic peptides and needs more sophisticated computational methods. Many uses of this resource can be envisaged ranging from the study of gene/protein expression regulation to protein biomarker specificity evaluation to name a few.

systems biology↗