| 1. |
- Wållberg, Helena, et al.
(författare)
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Affinity recovery of eight HER2-binding affibody variants using an anti-idiotypic affibody molecule as capture ligand
- 2011
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Ingår i: Protein Expression and Purification. - : Elsevier BV. - 1046-5928 .- 1096-0279. ; 76:1, s. 127-135
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Tidskriftsartikel (refereegranskat)abstract
- Affibody molecules generated by combinatorial protein engineering to bind the human epidermal growth factor receptor 2 (HER2) have in earlier studies proven to be promising tracers for HER2-mediated molecular imaging of cancer. Amino acid extensions either at the N- or C-terminus of these Z(HER2) affibody molecules, have been successfully employed for site-specific radiolabeling of the tracer candidates. Hexahistidyls or other tags, which would be convenient for recovery purposes, should be avoided since they could negatively influence the tumor targeting efficacy and biodistribution properties of the tracer. Using a new beta-lactamase-based protein fragment complementation assay (PCA), an affibody molecule was isolated which bound a Z(HER2) affibody molecule with sub-micromolar affinity, but not unrelated affibody molecules. This suggests that the interacting area include the HER2-binding surface of Z(HER2). This novel anti-idiotypic affibody molecule Z(E01) was produced in Escherichia coli, purified, and chemically coupled to a chromatography resin in order to generate an affibody-based affinity column, suitable for recovery of different variants of Z(HER2) affibody molecules, having a common binding surface for HER2. Eight such Z(HER2) affibody molecules, designed for future radioimaging investigations, having different C-terminal peptide extensions aimed for radioisotope (Tc-99m)-chelation, were successfully produced and recovered in a single step to high purity using the anti-idiotypic affibody ligand for the affinity purification. These results clearly suggest a potential for the development of anti-idiotypic affibody-based resins for efficient recovery of related variants of a target protein that might have altered biochemical properties, thus avoiding the cumbersome design of specific recovery schemes for each variant of a target protein.
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| 2. |
- Johansson, Maria U, et al.
(författare)
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Structure, specificity, and mode of interaction for bacterial albumin-binding modules.
- 2002
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Ingår i: Journal of Biological Chemistry. - 1083-351X .- 0021-9258. ; 277:10, s. 8114-8120
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Tidskriftsartikel (refereegranskat)abstract
- We have determined the solution structure of an albumin binding domain of protein G, a surface protein of group C and G streptococci. We find that it folds into a left handed three-helix bundle similar to the albumin binding domain of protein PAB from Peptostreptococcus magnus. The two domains share 59% sequence identity, are thermally very stable, and bind to the same site on human serum albumin. The albumin binding site, the first determined for this structural motif known as the GA module, comprises residues spanning the first loop to the beginning of the third helix and includes the most conserved region of GA modules. The two GA modules have different affinities for albumin from different species, and their albumin binding patterns correspond directly to the host specificity of C/G streptococci and P. magnus, respectively. These studies of the evolution, structure, and binding properties of the GA module emphasize the power of bacterial adaptation and underline ecological and medical problems connected with the use of antibiotics.
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| 3. |
- Alvez, Maria Bueno, et al.
(författare)
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Next generation pan-cancer blood proteome profiling using proximity extension assay
- 2023
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Ingår i: Nature Communications. - : Springer Nature. - 2041-1723. ; 14:1
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Tidskriftsartikel (refereegranskat)abstract
- A comprehensive characterization of blood proteome profiles in cancer patients can contribute to a better understanding of the disease etiology, resulting in earlier diagnosis, risk stratification and better monitoring of the different cancer subtypes. Here, we describe the use of next generation protein profiling to explore the proteome signature in blood across patients representing many of the major cancer types. Plasma profiles of 1463 proteins from more than 1400 cancer patients are measured in minute amounts of blood collected at the time of diagnosis and before treatment. An open access Disease Blood Atlas resource allows the exploration of the individual protein profiles in blood collected from the individual cancer patients. We also present studies in which classification models based on machine learning have been used for the identification of a set of proteins associated with each of the analyzed cancers. The implication for cancer precision medicine of next generation plasma profiling is discussed.
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| 4. |
- Fagerberg, Linn, et al.
(författare)
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Contribution of antibody-based protein profiling to the human chromosome-centric proteome project (C-HPP)
- 2013
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 12:6, s. 2439-2448
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Tidskriftsartikel (refereegranskat)abstract
- A gene-centric Human Proteome Project has been proposed to characterize the human protein-coding genes in a chromosome-centered manner to understand human biology and disease. Here, we report on the protein evidence for all genes predicted from the genome sequence based on manual annotation from literature (UniProt), antibody-based profiling in cells, tissues and organs and analysis of the transcript profiles using next generation sequencing in human cell lines of different origins. We estimate that there is good evidence for protein existence for 69% (n = 13985) of the human protein-coding genes, while 23% have only evidence on the RNA level and 7% still lack experimental evidence. Analysis of the expression patterns shows few tissue-specific proteins and approximately half of the genes expressed in all the analyzed cells. The status for each gene with regards to protein evidence is visualized in a chromosome-centric manner as part of a new version of the Human Protein Atlas (www.proteinatlas.org).
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| 5. |
- Niklasson, Mia, et al.
(författare)
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Membrane-Depolarizing Channel Blockers Induce Selective Glioma Cell Death by Impairing Nutrient Transport and Unfolded Protein/Amino Acid Responses
- 2017
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Ingår i: Cancer Research. - : AMER ASSOC CANCER RESEARCH. - 0008-5472 .- 1538-7445. ; 77:7, s. 1741-1752
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Tidskriftsartikel (refereegranskat)abstract
- Glioma-initiating cells (GIC) are considered the underlying cause of recurrences of aggressive glioblastomas, replenishing the tumor population and undermining the efficacy of conventional chemotherapy. Here we report the discovery that inhibiting T-type voltage-gated Ca2+ and KCa channels can effectively induce selective cell death of GIC and increase host survival in an orthotopic mouse model of human glioma. At present, the precise cellular pathways affected by the drugs affecting these channels are unknown. However, using cell-based assays and integrated proteomics, phosphoproteomics, and transcriptomics analyses, we identified the downstreamsignaling events these drugs affect. Changes in plasma membrane depolarization and elevated intracellular Na+, which compromised Na+-dependent nutrient transport, were documented. Deficits in nutrient deficit acted in turn to trigger the unfolded protein response and the amino acid response, leading ultimately to nutrient starvation and GIC cell death. Our results suggest new therapeutic targets to attack aggressive gliomas.
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| 6. |
- Sjöstedt, Evelina, et al.
(författare)
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Integration of Transcriptomics and Antibody-Based Proteomics for Exploration of Proteins Expressed in Specialized Tissues
- 2018
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 17:12, s. 4127-4137
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Tidskriftsartikel (refereegranskat)abstract
- A large portion of human proteins are referred to as missing proteins, defined as protein-coding genes that lack experimental data on the protein level due to factors such as temporal expression, expression in tissues that are difficult to sample, or they actually do not encode functional proteins. In the present investigation, an integrated omics approach was used for identification and exploration of missing proteins. Transcriptomics data from three different sourcesthe Human Protein Atlas (HPA), the GTEx consortium, and the FANTOM5 consortiumwere used as a starting point to identify genes selectively expressed in specialized tissues. Complementing the analysis with profiling on more specific tissues based on immunohistochemistry allowed for further exploration of cell-type-specific expression patterns. More detailed tissue profiling was performed for >300 genes on complementing tissues. The analysis identified tissue-specific expression of nine proteins previously listed as missing proteins (POU4F1, FRMD1, ARHGEF33, GABRG1, KRTAP2-1, BHLHE22, SPRR4, AVPR1B, and DCLK3), as well as numerous proteins with evidence of existence on the protein level that previously lacked information on spatial resolution and cell-type- specific expression pattern. We here present a comprehensive strategy for identification of missing proteins by combining transcriptomics with antibody-based proteomics. The analyzed proteins provide interesting targets for organ-specific research in health and disease.
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| 7. |
- Stahl, Stefan, et al.
(författare)
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Recombinant expression of insulin C-peptide
- 1997
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Patent (populärvet., debatt m.m.)abstract
- The invention relates to a method for the preparation of insulin C-peptide which includes expressing in a host cell of a multimer polypeptide containing copies of the insulin C-peptide, and degradation of the expressed polypeptide for the release of individual copies of the insulin C-peptide. The invention also relates to molecules of the nucleinic acid, to vectors of expression and host cells for the application of this method and to multimer insulin C-polypeptide expressed and degraded in accordance with the method. 21 claims
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| 8. |
- Uhlén, Mathias, et al.
(författare)
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A pathology atlas of the human cancer transcriptome
- 2017
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Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 357:6352, s. 660-
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Tidskriftsartikel (refereegranskat)abstract
- Cancer is one of the leading causes of death, and there is great interest in understanding the underlying molecular mechanisms involved in the pathogenesis and progression of individual tumors. We used systems-level approaches to analyze the genome-wide transcriptome of the protein-coding genes of 17 major cancer types with respect to clinical outcome. A general pattern emerged: Shorter patient survival was associated with up-regulation of genes involved in cell growth and with down-regulation of genes involved in cellular differentiation. Using genome-scale metabolic models, we show that cancer patients have widespread metabolic heterogeneity, highlighting the need for precise and personalized medicine for cancer treatment. All data are presented in an interactive open-access database (www.proteinatlas.org/pathology) to allow genome-wide exploration of the impact of individual proteins on clinical outcomes.
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| 9. |
- Uhlén, Mathias, et al.
(författare)
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Tissue-based map of the human proteome
- 2015
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Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 347:6220, s. 1260419-
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Tidskriftsartikel (refereegranskat)abstract
- Resolving the molecular details of proteome variation in the different tissues and organs of the human body will greatly increase our knowledge of human biology and disease. Here, we present a map of the human tissue proteome based on an integrated omics approach that involves quantitative transcriptomics at the tissue and organ level, combined with tissue microarray-based immunohistochemistry, to achieve spatial localization of proteins down to the single-cell level. Our tissue-based analysis detected more than 90% of the putative protein-coding genes. We used this approach to explore the human secretome, the membrane proteome, the druggable proteome, the cancer proteome, and the metabolic functions in 32 different tissues and organs. All the data are integrated in an interactive Web-based database that allows exploration of individual proteins, as well as navigation of global expression patterns, in all major tissues and organs in the human body.
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| 10. |
- Abdellah, Tebani, et al.
(författare)
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Integration of molecular profiles in a longitudinal wellness profiling cohort.
- 2020
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Ingår i: Nature communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 11:1
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Tidskriftsartikel (refereegranskat)abstract
- An important aspect of precision medicine is to probe the stability in molecular profiles among healthy individuals over time. Here, we sample a longitudinal wellness cohort with 100 healthy individuals and analyze blood molecular profiles including proteomics, transcriptomics, lipidomics, metabolomics, autoantibodies andimmune cell profiling, complementedwith gut microbiota composition and routine clinical chemistry. Overall, our results show high variation between individuals across different molecular readouts, while the intra-individual baseline variation is low. The analyses show that each individual has a unique and stable plasma protein profile throughout the study period and that many individuals also show distinct profiles with regards to the other omics datasets, with strong underlying connections between the blood proteome and the clinical chemistry parameters. In conclusion, the results support an individual-based definition of health and show that comprehensive omics profiling in a longitudinal manner is a path forward for precision medicine.
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| 11. |
- Agaton, Charlotta, et al.
(författare)
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Gene expression analysis by signature pyrosequencing
- 2002
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Ingår i: Gene. - 0378-1119 .- 1879-0038. ; 289:1-2, s. 31-39
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Tidskriftsartikel (refereegranskat)abstract
- We describe a novel method for transcript profiling based on high-throughput parallel sequencing of signature tags using a non-gel-based microtiter plate format. The method relies on the identification of cDNA clones by pyrosequencing of the region corresponding to the 3'-end of the mRNA preceding the poly(A) tail. Simultaneously, the method can be used for gene discovery, since tags corresponding to unknown genes can be further characterized by extended sequencing. The protocol was validated using a model system for human atherosclerosis. Two 3'-tagged cDNA libraries, representing macrophages and foam cells, which are key components in the development of atherosclerotic plaques, were constructed using a solid phase approach. The libraries were analyzed by pyrosequencing, giving on average 25 bases. As a control, conventional expressed sequence tag (EST) sequencing using slab gel electrophoresis was performed. Homology searches were used to identify the genes corresponding to each tag. Comparisons with EST sequencing showed identical, unique matches in the majority of cases when the pyrosignature was at least 18 bases. A visualization tool was developed to facilitate differential analysis using a virtual chip format. The analysis resulted in identification of genes with possible relevance for development of atherosclerosis. The use of the method for automated massive parallel signature sequencing is discussed.
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| 12. |
- Ahlin, Gustav, 1977-, et al.
(författare)
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Endogenous Gene and Protein Expression of Drug Transporting Proteins in Cell Lines Routinely used in Drug Discovery Programs
- 2009
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Ingår i: Drug Metabolism And Disposition. - : American Society for Pharmacology & Experimental Therapeutics (ASPET). - 0090-9556 .- 1521-009X. ; 37:12, s. 2275-2283
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Tidskriftsartikel (refereegranskat)abstract
- The aim of this study was to investigate the gene and protein expression profiles of important drug transporting proteins in human cell lines commonly used for studies of drug transport mechanisms. Human cell lines used to transiently or stably express single transporters (HeLa, HEK293) and leukaemia cell lines used to study drug resistance by ABC-transporters (HL-60, K562) were investigated, and compared with organotypic cell lines (HepG2, Saos-2, Caco-2 and Caco-2 TC7). For gene expression studies, real-time PCR was used, while monospecific polyclonal antibodies were generated and used to investigate protein expression by immunohistochemistry. Thirty-six transporters were studied for gene expression and nine for protein expression. The antibodies were validated using expression patterns in human tissues. Finally, the function of one ubiquitously expressed transporter, MCT1; SLC16A1 was investigated using 14C-lactic acid as a substrate. In general, the adherent cell lines (HeLa, HEK293) displayed low transporter expression and the expression patterns were barely affected by transfection. The leukaemia cell lines (K562, HL-60) and Saos-2 also had low endogenous transporter expression, while the organotypic cell lines (HepG2 and Caco-2) showed higher expression of some transporters. Comparison of gene and protein expression profiles gave poor correlations, but better agreement was obtained for antibodies with a good validation score, indicating that antibody quality was a significant variable. Importantly, the monocarboxylic acid transporting protein MCT1 was significantly expressed in all, and functional in most of the cell lines, indicating that MCT1 may be a confounding factor when the transport of small anionic drugs is investigated.
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| 13. |
- Andersson, Gustav, et al.
(författare)
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Reduced expression of ezrin in urothelial bladder cancer signifies more advanced tumours and an impaired survival : validatory study of two independent patient cohorts
- 2014
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Ingår i: BMC Urology. - : BioMed Central (BMC). - 1471-2490. ; 14:1, s. 36-
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Tidskriftsartikel (refereegranskat)abstract
- Background: Reduced membranous expression of the cytoskeleton-associated protein ezrin has previously been demonstrated to correlate with tumour progression and poor prognosis in patients with T1G3 urothelial cell carcinoma of the bladder treated with non-maintenance Bacillus Calmette-Guerin (n = 92), and the associations with adverse clinicopathological factors have been validated in another, unselected, cohort (n = 104). In the present study, we examined the prognostic significance of ezrin expression in urothelial bladder cancer in a total number of 442 tumours from two independent patient cohorts. Methods: Immunohistochemical expression of ezrin was evaluated in tissue microarrays with tumours from one retrospective cohort of bladder cancer (n = 110; cohort I) and one population-based cohort (n = 342; cohort II). Classification regression tree analysis was applied for selection of prognostic cutoff. Kaplan-Meier analysis, log rank test and Cox regression proportional hazards' modeling were used to evaluate the impact of ezrin on 5-year overall survival (OS), disease-specific survival (DSS) and progression-free survival (PFS). Results: Ezrin expression could be evaluated in tumours from 100 and 342 cases, respectively. In both cohorts, reduced membranous ezrin expression was significantly associated with more advanced T-stage (p < 0.001), high grade tumours (p < 0.001), female sex (p = 0.040 and p = 0.013), and membranous expression of podocalyxin-like protein (p < 0.001 and p = 0.009). Moreover, reduced ezrin expression was associated with a significantly reduced 5-year OS in both cohorts (HR = 3.09 95% CI 1.71-5.58 and HR = 2.15(1.51-3.06), and with DSS in cohort II (HR = 2.77, 95% CI 1.78-4.31). This association also remained significant in adjusted analysis in Cohort I (HR1.99, 95% CI 1.05-3.77) but not in Cohort II. In pTa and pT1 tumours in cohort II, there was no significant association between ezrin expression and time to progression. Conclusions: The results from this study validate previous findings of reduced membranous ezrin expression in urothelial bladder cancer being associated with unfavourable clinicopathological characteristics and an impaired survival. The utility of ezrin as a prognostic biomarker in transurethral resection specimens merits further investigation.
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| 14. |
- Aperia, Anita, et al.
(författare)
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Mending Fences : Na,K-ATPase signaling via Ca2+ in the maintenance of epithelium integrity
- 2020
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Ingår i: Cell Calcium. - : Elsevier BV. - 0143-4160 .- 1532-1991. ; 88
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Tidskriftsartikel (refereegranskat)abstract
- Na,K-ATPase is a ubiquitous multifunctional protein that acts both as an ion pump and as a signal transducer. The signaling function is activated by ouabain in non-toxic concentrations. In epithelial cells the ouabain-bound Na,K-ATPase connects with the inositol 1,4,5-trisphosphate receptor via a short linear motif to activate low frequency Ca2+ oscillations. Within a couple of minutes this ouabain mediated signal has resulted in phosphorylation or dephosphorylation of 2580 phospho-sites. Proteins that control cell proliferation and cell adhesion and calmodulin regulated proteins are enriched among the ouabain phosphor-regulated proteins. The inositol 1,4,5-trisphosphate receptor and the stromal interaction molecule, which are both essential for the initiation of Ca2+ oscillations, belong to the ouabain phosphor-regulated proteins. Downstream effects of the ouabain-evoked Ca2+ signal in epithelial cells include interference with the intrinsic mitochondrial apoptotic process and stimulation of embryonic growth processes. The dual function of Na,K-ATPase as an ion pump and a signal transducer is now well established and evaluation of the physiological and pathophysiological consequences of this universal signal emerges as an urgent topic for future studies.
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| 15. |
- Bandmann, Nina, et al.
(författare)
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Genetic engineering of the Fusarium solani pisi lipase cutinase for enhanced partitioning in PEG-phosphate aqueous two-phase systems
- 2000
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Ingår i: Journal of Biotechnology. - 0168-1656 .- 1873-4863. ; 79:2, s. 161-172
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Tidskriftsartikel (refereegranskat)abstract
- The Fusarium solani pisi lipase cutinase has been genetically engineered to investigate the influence of C-terminal peptide extensions on the partitioning of the enzyme in PEG-salt based aqueous two-phase bioseparation systems. Seven different cutinase lipase variants were constructed containing various C-terminal peptide extensions including tryptophan rich peptide tags ((WP)(2) and (WP)(4)), positively ((RP)(4)) and negatively ((DP)(4)) charged tags as well as combined tags with tryptophan together with either positively ((WPR)(4)) or negatively ((WPD)(4)) charged amino acids. The modified cutinase variants were stably produced in Escherichia coli as secreted to the periplasm from which they were efficiently purified by IgG-affinity chromatography employing an introduced N-terminal IgG-binding ZZ affinity fusion partner present in all variants. Partitioning experiments performed in a PEG 4000/sodium phosphate aqueous two-phase system showed that for variants containing either (WP)(2) or (WP)(4) peptide extensions, 10- to 70-fold increases in the partitioning to the PEG rich top-phase were obtained, when compared to the wild type enzyme. An increased partitioning was also seen for cutinase variants tagged with both tryptophans and charged amino acids, whereas the effect of solely charged peptide extensions was relatively small. In addition, when performing partitioning experiments from cell disintegrates, the (WP)(4)-tagged cutinase showed a similarly high PEG-phase partitioning, indicating that the effect from the peptide tag was unaffected by the background of the host proteins. Taken together, the results show that the partitioning of the recombinantly produced cutinase model enzyme could be significantly improved by relatively minor genetic engineering and that the effects observed for purified proteins are retained also in an authentic whole cell disintegrate system. The results presented should be of general interest also for the improvement of the partitioning properties of other industrially interesting proteins including bulk enzymes.
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| 16. |
- Barbe, Laurent, et al.
(författare)
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Toward a confocal subcellular atlas of the human proteome
- 2008
-
Ingår i: Molecular and cellular proteomics. - 1535-9476 .- 1535-9484. ; 7:3, s. 499-508
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Tidskriftsartikel (refereegranskat)abstract
- Information on protein localization on the subcellular level is important to map and characterize the proteome and to better understand cellular functions of proteins. Here we report on a pilot study of 466 proteins in three human cell lines aimed to allow large scale confocal microscopy analysis using protein-specific antibodies. Approximately 3000 high resolution images were generated, and more than 80% of the analyzed proteins could be classified in one or multiple subcellular compartment(s). The localizations of the proteins showed, in many cases, good agreement with the Gene Ontology localization prediction model. This is the first large scale antibody-based study to localize proteins into subcellular compartments using antibodies and confocal microscopy. The results suggest that this approach might be a valuable tool in conjunction with predictive models for protein localization.
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| 17. |
- Berglund, Lisa, et al.
(författare)
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A genecentric Human Protein Atlas for expression profiles based on antibodies
- 2008
-
Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 7:10, s. 2019-2027
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Forskningsöversikt (refereegranskat)abstract
- An attractive path forward in proteomics is to experimentally annotate the human protein complement of the genome in a genecentric manner. Using antibodies, it might be possible to design protein-specific probes for a representative protein from every protein-coding gene and to subsequently use the antibodies for systematical analysis of cellular distribution and subcellular localization of proteins in normal and disease tissues. A new version (4.0) of the Human Protein Atlas has been developed in a genecentric manner with the inclusion of all human genes and splice variants predicted from genome efforts together with a visualization of each protein with characteristics such as predicted membrane regions, signal peptide, and protein domains and new plots showing the uniqueness (sequence similarity) of every fraction of each protein toward all other human proteins. The new version is based on tissue profiles generated from 6120 antibodies with more than five million immunohistochemistry-based images covering 5067 human genes, corresponding to approximately 25% of the human genome. Version 4.0 includes a putative list of members in various protein classes, both functional classes, such as kinases, transcription factors, G-protein-coupled receptors, etc., and project-related classes, such as candidate genes for cancer or cardiovascular diseases. The exact antigen sequence for the internally generated antibodies has also been released together with a visualization of the application-specific validation performed for each antibody, including a protein array assay, Western blot analysis, immunohistochemistry, and, for a large fraction, immunofluorescence-based confocal microscopy. New search functionalities have been added to allow complex queries regarding protein expression profiles, protein classes, and chromosome location. The new version of the protein atlas thus is a resource for many areas of biomedical research, including protein science and biomarker discovery.
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| 18. |
- Binz, Hans, et al.
(författare)
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Method for enhancing the immunogenicity of an immunogenic compound or hapten, and use thereof for preparing vaccines
- 1994
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Patent (populärvet., debatt m.m.)abstract
- The present invention relates to a process for improving the immunogenicity of an immunogen, an antigen or a hapten, when it is administered to a host, independently of the mode of administration, characterized in that the said antigen or hapten is coupled covalently to a support molecule in order to form a complex, and in that this support molecule is a polypeptide fragment which is able to bind specifically to mammalian serum albumin. The invention also relates to the use, as a medicament, of the product which can be obtained in this way.
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| 19. |
- Binz, Hans, et al.
(författare)
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Respiratory syncytial virus protein g expressed on bacterial membrane
- 1994
-
Patent (populärvet., debatt m.m.)abstract
- A method for preparing a peptide or protein, wherein (a) a DNA sequence coding for a heterologous polypeptide on a peptide sequence between amino acid residues 130 and 230 of respiratory syncytial virus protein G, sub-groups A and B, or a peptide sequence at least 80 % homologous thereto, and (b) means enabling the expression of the polypeptide on the bacterial membrane surface, are inserted into a bacterium which is not pathogenic for mammals. The resulting conjugate polypeptide and a live bacterium expressing same, pharmaceutical compositions containing them and their use for preparing a vaccine, as well as a DNA sequence coding for said polypeptide, are also disclosed.
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| 20. |
- Björling, Erik, et al.
(författare)
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A web-based tool for in silico biomarker discovery based on tissue-specific protein profiles in normal and cancer tissues
- 2008
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Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 7:5, s. 825-844
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Tidskriftsartikel (refereegranskat)abstract
- Here we report the development of a publicly available Web-based analysis tool for exploring proteins expressed in a tissue- or cancer-specific manner. The search queries are based on the human tissue profiles in normal and cancer cells in the Human Protein Atlas portal and rely on the individual annotation performed by pathologists of images representing immunohistochemically stained tissue sections. Approximately 1.8 million images representing more than 3000 antibodies directed toward human proteins were used in the study. The search tool allows for the systematic exploration of the protein atlas to discover potential protein biomarkers. Such biomarkers include tissue-specific markers, cell type-specific markers, tumor type-specific markers, markers of malignancy, and prognostic or predictive markers of cancers. Here we show examples of database queries to generate sets of candidate biomarker proteins for several of these different categories. Expression profiles of candidate proteins can then subsequently be validated by examination of the underlying high resolution images. The present study shows examples of search strategies revealing several potential protein biomarkers, including proteins specifically expressed in normal cells and in cancer cells from specified tumor types. The lists of candidate proteins can be used as a starting point for further validation in larger patient cohorts using both immunological approaches and technologies utilizing more classical proteomics tools.
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| 21. |
- Boman, Karolina, et al.
(författare)
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Decreased expression of RNA-binding motif protein 3 correlates with tumour progression and poor prognosis in urothelial bladder cancer
- 2013
-
Ingår i: BMC Urology. - : Springer Science and Business Media LLC. - 1471-2490. ; 13:April,8, s. 17-17
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Low nuclear expression of the RNA-binding motif protein 3 (RBM3) has previously been found to be associated with poor prognosis in several cancer forms e.g. breast, ovarian, colorectal, prostate cancer and malignant melanoma. The aim of this study was to examine the prognostic impact of RBM3 expression in urinary bladder cancer.METHODS: Immunohistochemical RBM3 expression was examined in tumours from 343 patients with urothelial bladder cancer. Chi-square and Spearman's correlation tests were applied to explore associations between RBM3 expression and clinicopathological characteristics. The impact of RBM3 expression on disease-specific survival (DSS), 5-year overall survival (OS) and progression-free survival (PFS) was assessed by Kaplan-Meier analysis and Cox proportional hazards modelling.RESULTS: Reduced nuclear RBM3 expression was significantly associated with more advanced tumour (T) stage (p <0.001) and high grade tumours (p=0.004). Negative RBM3 expression was associated with a significantly shorter DSS (HR=2.55; 95% CI 1.68-3.86)) and 5-year OS (HR=2.10; 95% CI 1.56-2.82), also in multivariable analysis (HR=1.65; 95% CI 1.07-2.53 for DSS and HR=1.54; 95% CI 1.13-2.10 for 5-year OS). In patients with Ta and T1 tumours expressing reduced RBM3 levels, Kaplan-Meier analysis revealed a significantly shorter PFS (p=0.048) and 5-year OS (p=0.006).CONCLUSION: Loss of RBM3 expression is associated with clinically more aggressive tumours and an independent factor of poor prognosis in patients with urothelial bladder cancer and a potentially useful biomarker for treatment stratification and surveillance of disease progression.
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| 22. |
- Boman, Karolina, et al.
(författare)
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Membranous expression of podocalyxin-like protein is an independent factor of poor prognosis in urothelial bladder cancer
- 2013
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Ingår i: British Journal of Cancer. - : Springer Science and Business Media LLC. - 0007-0920 .- 1532-1827. ; 108:11, s. 2321-2328
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Tidskriftsartikel (refereegranskat)abstract
- Background: Membranous expression of the anti-adhesive glycoprotein podocalyxin-like (PODXL) has previously been found to correlate with poor prognosis in several major cancer forms. Here we examined the prognostic impact of PODXL expression in urothelial bladder cancer. Methods: Immunohistochemical PODXL expression was examined in tissue microarrays with tumours from two independent cohorts of patients with urothelial bladder cancer: n = 100 (Cohort I) and n = 343 (Cohort II). The impact of PODXL expression on disease-specific survival (DSS; Cohort II), 5-year overall survival (OS; both cohorts) and 2-year progression-free survival (PFS; Cohort II) was assessed. Results: Membranous PODXL expression was significantly associated with more advanced tumour (T) stage and high-grade tumours in both cohorts, and a significantly reduced 5-year OS (unadjusted HR = 2.25 in Cohort I and 3.10 in Cohort II, adjusted HR = 2.05 in Cohort I and 2.18 in Cohort II) and DSS (unadjusted HR = 4.36, adjusted HR = 2.70). In patients with Ta and T1 tumours, membranous PODXL expression was an independent predictor of a reduced 2-year PFS (unadjusted HR = 6.19, adjusted HR = 4.60) and DSS (unadjusted HR = 8.34, adjusted HR = 7.16). Conclusion: Membranous PODXL expression is an independent risk factor for progressive disease and death in patients with urothelial bladder cancer.
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| 23. |
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| 24. |
- Brautigam, Lars, et al.
(författare)
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Glutaredoxin regulates vascular development by reversible glutathionylation of sirtuin 1
- 2013
-
Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 110:50, s. 20057-20062
-
Tidskriftsartikel (refereegranskat)abstract
- Embryonic development depends on complex and precisely orchestrated signaling pathways including specific reduction/oxidation cascades. Oxidoreductases of the thioredoxin family are key players conveying redox signals through reversible posttranslational modifications of protein thiols. The importance of this protein family during embryogenesis has recently been exemplified for glutaredoxin 2, a vertebrate-specific glutathione-disulfide oxidoreductase with a critical role for embryonic brain development. Here, we discovered an essential function of glutaredoxin 2 during vascular development. Confocal microscopy and time-lapse studies based on two-photon microscopy revealed that morpholino-based knockdown of glutaredoxin 2 in zebrafish, a model organism to study vertebrate embryogenesis, resulted in a delayed and disordered blood vessel network. We were able to show that formation of a functional vascular system requires glutaredoxin 2-dependent reversible S-glutathionylation of the NAD(+)-dependent protein deacetylase sirtuin 1. Using mass spectrometry, we identified a cysteine residue in the conserved catalytic region of sirtuin 1 as target for glutaredoxin 2-specific deglutathionylation. Thereby, glutaredoxin 2-mediated redox regulation controls enzymatic activity of sirtuin 1, a mechanism we found to be conserved between zebrafish and humans. These results link S-glutathionylation to vertebrate development and successful embryonic angiogenesis.
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| 25. |
- Byström, Sanna, et al.
(författare)
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Affinity Proteomics Exploration of Melanoma Identifies Proteins in Serum with Associations to T-Stage and Recurrence
- 2017
-
Ingår i: Translational Oncology. - : Neoplasia Press, Inc.. - 1944-7124 .- 1936-5233. ; 10:3, s. 385-395
-
Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Blood-based proteomic profiling may aid and expand our understanding of diseases and their different phenotypes. The aim of the presented study was to profile serum samples from patients with malignant melanoma using affinity proteomic assays to describe proteins in the blood stream that are associated to stage or recurrence of melanoma. MATERIAL AND METHODS: Multiplexed protein analysis was conducted using antibody suspension bead arrays. A total of 232 antibodies against 132 proteins were selected from (i) a screening with 4595 antibodies and 32 serum samples from melanoma patients and controls, (ii) antibodies used for immunohistochemistry, (iii) protein targets previously related with melanoma. The analysis was performed with 149 serum samples from patients with malignant melanoma. Antibody selectivity was then assessed by Western blot, immunocapture mass spectrometry, and epitope mapping. Lastly, indicative antibodies were applied for IHC analysis of melanoma tissues. RESULTS: Serum levels of regucalcin (RGN) and syntaxin 7 (STX7) were found to be lower in patients with both recurring tumors and a high Breslow's thickness (T-stage 3/4) compared to low thickness (T-stage 1/2) without disease recurrence. Serum levels of methylenetetrahydrofolate dehydrogenase 1-like (MTHFD1L) were instead elevated in sera of T3/4 patients with recurrence. The analysis of tissue sections with S100A6 and MTHFD1L showed positive staining in a majority of patients with melanoma, and S100A6 was significantly associated to T-stage. CONCLUSIONS: Our findings provide a starting point to further study RGN, STX7, MTHFD1L and S100A6 in serum to elucidate their involvement in melanoma progression and to assess a possible contribution to support clinical indications.
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| 26. |
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| 27. |
- Edfors, Fredrik, et al.
(författare)
-
Enhanced validation of antibodies for research applications
- 2018
-
Ingår i: Nature Communications. - : Nature Publishing Group. - 2041-1723. ; 9
-
Tidskriftsartikel (refereegranskat)abstract
- There is a need for standardized validation methods for antibody specificity and selectivity. Recently, five alternative validation pillars were proposed to explore the specificity of research antibodies using methods with no need for prior knowledge about the protein target. Here, we show that these principles can be used in a streamlined manner for enhanced validation of research antibodies in Western blot applications. More than 6,000 antibodies were validated with at least one of these strategies involving orthogonal methods, genetic knockdown, recombinant expression, independent antibodies, and capture mass spectrometry analysis. The results show a path forward for efforts to validate antibodies in an application-specific manner suitable for both providers and users.
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| 28. |
- Edqvist, Per-Henrik D, et al.
(författare)
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Expression of Human Skin-Specific Genes Defined by Transcriptomics and Antibody-Based Profiling
- 2015
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Ingår i: Journal of Histochemistry and Cytochemistry. - : SAGE Publications. - 0022-1554 .- 1551-5044. ; 63:2, s. 129-141
-
Tidskriftsartikel (refereegranskat)abstract
- To increase our understanding of skin, it is important to define the molecular constituents of the cell types and epidermal layers that signify normal skin. We have combined a genome-wide transcriptomics analysis, using deep sequencing of mRNA from skin biopsies, with immunohistochemistry-based protein profiling to characterize the landscape of gene and protein expression in normal human skin. The transcriptomics and protein expression data of skin were compared to 26 (RNA) and 44 (protein) other normal tissue types. All 20,050 putative protein-coding genes were classified into categories based on patterns of expression. We found that 417 genes showed elevated expression in skin, with 106 genes expressed at least five-fold higher than that in other tissues. The 106 genes categorized as skin enriched encoded for well-known proteins involved in epidermal differentiation and proteins with unknown functions and expression patterns in skin, including the C1orf68 protein, which showed the highest relative enrichment in skin. In conclusion, we have applied a genome-wide analysis to identify the human skin-specific proteome and map the precise localization of the corresponding proteins in different compartments of the skin, to facilitate further functional studies to explore the molecular repertoire of normal skin and to identify biomarkers related to various skin diseases.
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| 29. |
- Edqvist, Per-Henrik D., et al.
(författare)
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Loss of ASRGL1 expression is an independent biomarker for disease-specific survival in endometrioid endometrial carcinoma
- 2015
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Ingår i: Gynecologic Oncology. - : Elsevier BV. - 0090-8258 .- 1095-6859. ; 137:3, s. 529-537
-
Tidskriftsartikel (refereegranskat)abstract
- Objective For endometrial carcinoma, prognostic stratification methods do not satisfactorily identify patients with adverse outcome. Currently, histology, tumor grade and stage are used to tailoring surgical treatment and to determine the need for adjuvant treatment. Low-risk patients are not considered to require adjuvant therapy or staging lymphadenectomy. For patients with intermediate or high risk, some guidelines recommend tailoring adjuvant treatment according to additional negative prognostic factors. Our objective was to evaluate the biomarker potential of the ASRGL1 protein in endometrial carcinoma. Methods Using The Human Protein Atlas (www.proteinatlas.org), the l-asparaginase (ASRGL1) protein was identified as an endometrial carcinoma biomarker candidate. ASRGL1 expression was immunohistochemically evaluated with an extensively validated antibody on two independent endometrial carcinoma cohorts (n = 229 and n = 286) arranged as tissue microarrays. Staining results were correlated with clinical features. Results Reduced expression of ASRGL1, defined as < 75% positively stained tumor cells, was significantly associated with poor prognosis and reduced disease-specific survival in endometrioid endometrial adenocarcinoma (EEA). In multivariate analysis the hazard ratios for disease-specific survival were 3.55 (95% CI = 1.10-11.43; p = 0.003) and 3.23 (95% CI = 1.53-6.81; p = 0.002) in the two cohorts, respectively. Of the 48 cases with Grade 3 Stage I tumor all disease-related deaths were associated with low ASRGL1 expression. Conclusions Loss of ASRGL1 in EEA is a powerful biomarker for poor prognosis and retained ASRGL1 has a positive impact on survival. ASRGL1 immunohistochemistry has potential to become an additional tool for prognostication in cases where tailoring adjuvant treatment according to additional prognostic factors besides grade and stage is recommended.
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| 30. |
- Eklund, M., et al.
(författare)
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Anti-idiotypic protein domains selected from protein A-based affibody libraries
- 2002
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Ingår i: Proteins. - : Wiley. - 0887-3585 .- 1097-0134. ; 48:3, s. 454-462
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Tidskriftsartikel (refereegranskat)abstract
- Three pairs of small protein domains showing binding behavior in analogy with anti-idiotypic antibodies have been selected using phage display technology. From an affibody protein library constructed by combinatorial variegation of the Fe binding surface of the 58 residue staphylococcal protein A (SPA)-derived domain Z, affibody variants have been selected to the parental SPA scaffold and to two earlier identified SPA-derived affibodies. One selected affibody (Z(SPA-1)) was shown to recognize each of the five domains of wild-type SPA with dissociation constants (K.) in the micromolar range. The binding of the Z(SPA-1) affibody to its parental structure was shown to involve the Fc binding site of SPA, while the Fab-binding site was not involved. Similarly, affibodies showing anti-idiotypic binding characteristics were also obtained when affibodies previously selected for binding to Taq DNA polymerase and human IgA, respectively, were used as targets for selections. The potential applications for these types of affinity pairs were exemplified by one-step protein recovery using affinity chromatography employing the specific interactions between the respective protein pair members. These experiments included the purification of the Z(SPA-1) affibody from a total Escherichia coli cell lysate using protein A-Sepharose, suggesting that this protein A/antiprotein A affinity pair could provide a basis for novel affinity gene fusion systems. The use of this type of small, robust, and easily expressed anti-idiotypic affibody pair for affinity technology applications, including self-assembled protein networks, is discussed.
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| 31. |
- Eriksson, Hanna, et al.
(författare)
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Quantitative membrane proteomics applying narrow range peptide isoelectric focusing for studies of small cell lung cancer resistance mechanisms
- 2008
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Ingår i: Proteomics. - : Wiley. - 1615-9853 .- 1615-9861. ; 28:5C, s. 3275-3276
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Tidskriftsartikel (refereegranskat)abstract
- Drug resistance is often associated with upregulation of membrane-associated drug-efflux systems, and thus global membrane proteomics methods are valuable tools in the search for novel components of drug resistance phenotypes. Herein we have compared the microsomal proteome from the lung cancer cell line H69 and its isogenic Doxorubicin-resistant subcell line H69AR. The method used includes microsome preparation, iTRAQ labeling followed by narrow range peptide IEF in an immobilized pH-gradient (IPG-IEF) and LC-MS/MS analysis. We demonstrate that the microsomal preparation and iTRAQ labeling is reproducible regarding protein content and composition. The rationale using narrow range peptide IPG-IEF separation is demonstrated by its ability to: (i) lowering the complexity of the sample by two-thirds while keeping high proteome coverage (96%), (ii) providing high separation efficiency, and (iii) allowing for peptide validation and possibly identifications of post-transcriptional modifications. After analyzing one-fifth of the IEF fractions (effective pH range of 4.0-4.5), a total of 3704 proteins were identified, among which 527 were predicted to be membrane proteins. One of the proteins found to be differentially expressed was Serca 2, a calcium pump located in the ER membrane that potentially could result in changes of apoptotic response toward Doxorubicin.
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| 32. |
- Fagerberg, Linn, et al.
(författare)
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Analysis of the human tissue-specific expression by genome-wide integration of transcriptomics and antibody-based proteomics
- 2014
-
Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 13:2, s. 397-406
-
Tidskriftsartikel (refereegranskat)abstract
- Global classification of the human proteins with regards to spatial expression patterns across organs and tissues is important for studies of human biology and disease. Here, we used a quantitative transcriptomics analysis (RNA-Seq) to classify the tissue-specific expression of genes across a representative set of all major human organs and tissues and combined this analysis with antibody- based profiling of the same tissues. To present the data, we launch a new version of the Human Protein Atlas that integrates RNA and protein expression data corresponding to 80% of the human protein-coding genes with access to the primary data for both the RNA and the protein analysis on an individual gene level. We present a classification of all human protein-coding genes with regards to tissue-specificity and spatial expression pattern. The integrative human expression map can be used as a starting point to explore the molecular constituents of the human body.
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| 33. |
- Forsberg, David, et al.
(författare)
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CO2-evoked release of PGE2 modulates sighs and inspiration as demonstrated in brainstem organotypic culture.
- 2016
-
Ingår i: eLife. - 2050-084X. ; 5
-
Tidskriftsartikel (refereegranskat)abstract
- Inflammation-induced release of prostaglandin E2 (PGE2) changes breathing patterns and the response to CO2 levels. This may have fatal consequences in newborn babies and result in sudden infant death. To elucidate the underlying mechanisms, we present a novel breathing brainstem organotypic culture that generates rhythmic neural network and motor activity for 3 weeks. We show that increased CO2 elicits a gap junction-dependent release of PGE2. This alters neural network activity in the preBötzinger rhythm-generating complex and in the chemosensitive brainstem respiratory regions, thereby increasing sigh frequency and the depth of inspiration. We used mice lacking eicosanoid prostanoid 3 receptors (EP3R), breathing brainstem organotypic slices and optogenetic inhibition of EP3R(+/+) cells to demonstrate that the EP3R is important for the ventilatory response to hypercapnia. Our study identifies a novel pathway linking the inflammatory and respiratory systems, with implications for inspiration and sighs throughout life, and the ability to autoresuscitate when breathing fails.
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| 34. |
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| 35. |
- Grapotte, Mathys, et al.
(författare)
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Discovery of widespread transcription initiation at microsatellites predictable by sequence-based deep neural network
- 2021
-
Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723.
-
Tidskriftsartikel (refereegranskat)abstract
- Using the Cap Analysis of Gene Expression (CAGE) technology, the FANTOM5 consortium provided one of the most comprehensive maps of transcription start sites (TSSs) in several species. Strikingly, ~72% of them could not be assigned to a specific gene and initiate at unconventional regions, outside promoters or enhancers. Here, we probe these unassigned TSSs and show that, in all species studied, a significant fraction of CAGE peaks initiate at microsatellites, also called short tandem repeats (STRs). To confirm this transcription, we develop Cap Trap RNA-seq, a technology which combines cap trapping and long read MinION sequencing. We train sequence-based deep learning models able to predict CAGE signal at STRs with high accuracy. These models unveil the importance of STR surrounding sequences not only to distinguish STR classes, but also to predict the level of transcription initiation. Importantly, genetic variants linked to human diseases are preferentially found at STRs with high transcription initiation level, supporting the biological and clinical relevance of transcription initiation at STRs. Together, our results extend the repertoire of non-coding transcription associated with DNA tandem repeats and complexify STR polymorphism.
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| 36. |
- Grapotte, M, et al.
(författare)
-
Discovery of widespread transcription initiation at microsatellites predictable by sequence-based deep neural network
- 2021
-
Ingår i: Nature communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 12:1, s. 3297-
-
Tidskriftsartikel (refereegranskat)abstract
- Using the Cap Analysis of Gene Expression (CAGE) technology, the FANTOM5 consortium provided one of the most comprehensive maps of transcription start sites (TSSs) in several species. Strikingly, ~72% of them could not be assigned to a specific gene and initiate at unconventional regions, outside promoters or enhancers. Here, we probe these unassigned TSSs and show that, in all species studied, a significant fraction of CAGE peaks initiate at microsatellites, also called short tandem repeats (STRs). To confirm this transcription, we develop Cap Trap RNA-seq, a technology which combines cap trapping and long read MinION sequencing. We train sequence-based deep learning models able to predict CAGE signal at STRs with high accuracy. These models unveil the importance of STR surrounding sequences not only to distinguish STR classes, but also to predict the level of transcription initiation. Importantly, genetic variants linked to human diseases are preferentially found at STRs with high transcription initiation level, supporting the biological and clinical relevance of transcription initiation at STRs. Together, our results extend the repertoire of non-coding transcription associated with DNA tandem repeats and complexify STR polymorphism.
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| 37. |
- Gremel, Gabriela, et al.
(författare)
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A systematic analysis of commonly used antibodies in cancer diagnostics
- 2014
-
Ingår i: Histopathology. - : Wiley. - 0309-0167 .- 1365-2559. ; 64:2, s. 293-305
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Tidskriftsartikel (refereegranskat)abstract
- AimsImmunohistochemistry plays a pivotal role in cancer differential diagnostics. To identify the primary tumour from a metastasis specimen remains a significant challenge, despite the availability of an increasing number of antibodies. The aim of the present study was to provide evidence-based data on the diagnostic power of antibodies used frequently for clinical differential diagnostics. Methods and resultsA tissue microarray cohort comprising 940 tumour samples, of which 502 were metastatic lesions, representing tumours from 18 different organs and four non-localized cancer types, was analysed using immunohistochemistry with 27 well-established antibodies used in clinical differential diagnostics. Few antibodies, e.g. prostate-specific antigen and thyroglobulin, showed a cancer type-related sensitivity and specificity of more than 95%. A majority of the antibodies showed a low degree of sensitivity and specificity for defined cancer types. Combinations of antibodies provided limited added value for differential diagnostics of cancer types. ConclusionsThe results from analysing 27 diagnostic antibodies on consecutive sections of 940 defined tumours provide a unique repository of data that can empower a more optimal use of clinical immunohistochemistry. Our results highlight the benefit of immunohistochemistry and the unmet need for novel markers to improve differential diagnostics of cancer.
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| 38. |
- Gremel, Gabriela, et al.
(författare)
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A systematic search strategy identifies cubilin as independent prognostic marker for renal cell carcinoma
- 2017
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Ingår i: BMC Cancer. - : Springer Science and Business Media LLC. - 1471-2407. ; 17
-
Tidskriftsartikel (refereegranskat)abstract
- Background: There is an unmet clinical need for better prognostic and diagnostic tools for renal cell carcinoma (RCC). Methods: Human Protein Atlas data resources, including the transcriptomes and proteomes of normal and malignant human tissues, were searched for RCC-specific proteins and cubilin (CUBN) identified as a candidate. Patient tissue representing various cancer types was constructed into a tissue microarray (n = 940) and immunohistochemistry used to investigate the specificity of CUBN expression in RCC as compared to other cancers. Two independent RCC cohorts (n = 181; n = 114) were analyzed to further establish the sensitivity of CUBN as RCC-specific marker and to explore if the fraction of RCCs lacking CUBN expression could predict differences in patient survival. Results: CUBN was identified as highly RCC-specific protein with 58% of all primary RCCs staining positive for CUBN using immunohistochemistry. In venous tumor thrombi and metastatic lesions, the frequency of CUBN expression was increasingly lost. Clear cell RCC (ccRCC) patients with CUBN positive tumors had a significantly better prognosis compared to patients with CUBN negative tumors, independent of T-stage, Fuhrman grade and nodal status (HR 0.382, CI 0.203-0.719, P = 0.003). Conclusions: CUBN expression is highly specific to RCC and loss of the protein is significantly and independently associated with poor prognosis. CUBN expression in ccRCC provides a promising positive prognostic indicator for patients with ccRCC. The high specificity of CUBN expression in RCC also suggests a role as a new diagnostic marker in clinical cancer differential diagnostics to confirm or rule out RCC.
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| 39. |
- Gry, Marcus, et al.
(författare)
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Tissue-specific protein expression in human cells, tissues and organs
- 2010
-
Ingår i: Journal of Proteomics and Bioinformatics. - : OMICS Publishing Group. - 0974-276X. ; 3:10, s. 286-293
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Tidskriftsartikel (refereegranskat)abstract
- An important part of understanding human biology is the study of tissue-specific expression both at the gene and protein level. In this study, the analysis of tissue specific protein expression was performed based on tissue micro array data available on the public Human Protein Atlas database (www.proteinatlas.org). An analysis of human proteins, corresponding to approximately one third of the protein-encoding genes, was carried out in 65 human tissues and cell types. The spatial distribution and relative abundance of 6,678 human proteins, were analyzed in different cell populations from various organs and tissues in the human body using unsupervised methods, such as hierarchical clustering and principal component analysis, as well as with supervised methods (Breiman, 2001). Well-known markers, such as neuromodulin for the central nervous system, keratin 20 for gastrointestinal tract and CD45 for hematopoietic cells, were identified as tissue-specific. Proteins expressed in a tissue-specific manner were identified for cells in all of the investigated tissues, including the central nervous system, hematopoietic system, squamous epithelium, mesenchymal cells and cells from the gastrointestinal tract. Several proteins not yet associated with tissue-specificity were identified, providing starting points for further studies to explore tissue-specific functions. This includes proteins with no known function, such as ZNF509 expressed in CNS and C1orf201 expressed in the gastro-intestinal tract. In general, the majority of the gene products are expressed in a ubiquitous manner and few proteins are detected exclusively in cells from a particular tissue class, as exemplified by less than 1% of the analyzed proteins found only in the brain.
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| 40. |
- Gräslund, Susanne, et al.
(författare)
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A novel affinity gene fusion system allowing protein A-based recovery of non-immunoglobulin gene products
- 2002
-
Ingår i: Journal of Biotechnology. - 0168-1656 .- 1873-4863. ; 99:1, s. 41-50
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Tidskriftsartikel (refereegranskat)abstract
- An expression vector system has been developed, taking advantage of a novel, Staphylococcus aureus protein A (SPA)-binding affinity tag ZSPA-1, enabling straightforward affinity blotting procedures and efficient recovery by affinity purification of expressed gene products on readily available reagents and chromatography media. The 58 amino acid SPA-binding affinity tag ZSPA-1, was previously selected from a library constructed by combinatorial mutagenesis of a protein domain from SPA. An Escherichia coli expression vector for intracellular T7 promoter (PT7) driven production was constructed with an N-terminal dual affinity tag, consisting of a hexahistidyl (His6) tag in frame with the ZSPA-1 tag, thus allowing alternative affinity recovery methods. To evaluate the system, five cDNA clones from a mouse testis cDNA library were expressed, and two alternative blotting procedures were developed for convenient screening of expression efficiencies. The five produced fusion proteins were recovered on both immobilized metal-ion affinity chromatography (IMAC) columns and on Protein A-based chromatography media, to allow comparative studies. It was found that the Protein A-based recovery resulted in the highest degree of purity, and furthermore, gene products that were produced as inclusion bodies could after denaturation be efficiently affinity purified on Protein A-Sepharose in the presence of 0.5 M guanidine hydrochloride. The convenience and robustness of the presented expression system should make it highly suitable for various high-throughput protein expression efforts.
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| 41. |
- Gräslund, Torbjörn, et al.
(författare)
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Charge engineering of a protein domain to allow efficient ion-exchange recovery
- 2000
-
Ingår i: Protein Engineering. - : Oxford University Press (OUP). - 0269-2139 .- 1460-213X. ; 13:10, s. 703-709
-
Tidskriftsartikel (refereegranskat)abstract
- We have created protein domains with extreme surface charge. These mutated domains allow for ion-exchange chromatography under conditions favourable for selective and efficient capture, using Escherichia coli as a host organism. The staphylococcal protein A-derived domain Z (Z(wt)) was used asa scaffold when constructing two mutants, Z(basic1) and Z(basic2), with high positive surface charge. Far-ultraviolet circular dichroism measurements showed that they have a secondary structure content comparable to the parental molecule Z(wt). Although melting temperatures (T-m) of the engineered domains were lower than that of the wild-type Z domain, both mutants could be produced successfully as intracellular full-length products in E. coli and purified to homogeneity by ion-exchange chromatography. Further studies performed on Z(basic1) and Z(basic2) showed that they were able to bind to a cation exchanger even at pH values in the 9 to 11 range. A gene fusion between Z(basic2) and the acidic human serum albumin binding domain (ABD), derived from streptococcal protein G, was also constructed. The gene product Z(basic2)-ABD could be purified using cation-exchange chromatography from a whole cell lysate to more than 90% purity.
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| 42. |
- Gräslund, Torbjörn, et al.
(författare)
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Integrated strategy for selective expanded bed ion-exchange adsorption and site-specific protein processing using gene fusion technology
- 2002
-
Ingår i: Journal of Biotechnology. - : Elsevier BV. - 0168-1656 .- 1873-4863. ; 96:1, s. 93-102
-
Tidskriftsartikel (refereegranskat)abstract
- The highly charged domain Z(basic) can be used as a fusion partner to enhance adsorption of target proteins to cation exchanging resins at high pH-values. In this paper, we describe a strategy for purification of target proteins fused to Z(basic) at a constant physiological pH using cation exchange chromatography in an expanded bed mode. We show that two proteins, Klenow DNA polymerase and the viral protease 3C, can be efficiently purified from unclarified Escherichia coli homogenates in a single step with a selectivity analogous to what is normally achieved by affinity chromatography. The strategy also includes an integrated site-specific removal of the Z(basic) purification handle to yield a free target protein.
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| 43. |
- Gräslund, Torbjörn, et al.
(författare)
-
Production of a Thermostable DNA Polymerase by Site-Specific Cleavage of a Heat-Eluted Affinity Fusion Protein
- 1997
-
Ingår i: Protein Expression and Purification. - : Elsevier BV. - 1046-5928 .- 1096-0279. ; 9, s. 125-132
-
Tidskriftsartikel (refereegranskat)abstract
- A novel strategy is described for bacterial expression and affinity purification of a recombinant truncated version of the heat-stable DNA polymerase I fromThermus aquaticus.The DNA polymerase ([Delta]Taq) was produced as a fusion to a serum albumin binding affinity handle (ABP) derived from streptococcal protein G. Based on the thermostability of the [Delta]TaqDNA polymerase, affinity-purified ABP-[Delta]Taqcould be heat-eluted from HSA columns by incubation at 85ï¿œC. To produce free [Delta]TaqDNA polymerase, efficient site-specific cleavage of the affinity tag was performed using a recombinant coxsackievirus 3C protease (3Cpro), also produced as an ABP affinity fusion. Thus, an integrated strategy could be devised where both the cleaved ABP affinity tag and the protease fusion could be recovered after site-specific cleavage using HSA-affinity chromatography. The flow-through fraction contained essentially pure [Delta]TaqDNA polymerase with full enzymatic activity.
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| 44. |
- Gräslund, Torbjörn, et al.
(författare)
-
Strategy for highly selective ion-exchange capture using a charge-polarizing fusion partner
- 2002
-
Ingår i: Journal of Chromatography A. - 0021-9673 .- 1873-3778. ; 942:1-2, s. 157-166
-
Tidskriftsartikel (refereegranskat)abstract
- To achieve efficient recovery of recombinantly produced target proteins using cation-exchange chromatography, a novel basic protein domain is used as a purification handle. The proteolytic instability usually encountered for basic peptide tags is avoided by the use of a highly constrained α-helical domain based on staphylococcal protein A into which positively charged amino acids have been introduced. Here we show that this domain, consisting of 58 amino acids with a calculated isoelectric point (pI) of 10.5, can be used to efficiently capture different fused target proteins, such as a bacterial DNA polymerase (Klenow fragment), a viral protease (3C) and a fungal lipase (Cutinase). In contrast to standard cation-exchange chromatography, efficient capture can be achieved also at a pH value higher than the pI of the fusion protein, demonstrated here by Zbasic-Klenow polymerase (pI≈5.8) and ZZ-Cutinase-Zbasic (pI≈7.2) both purified at a pH of 7.5. These results show that the Zbasic domain is able to confer a regional concentration of positive charge on the fusion protein even at a relatively high pH. Hence, the data suggest that this domain could be used for highly efficient and selective capture of target proteins at conditions where most host-cell proteins do not bind to the chromatographic resin. The obtained purity after this one-step procedure suggests that the strategy could be an alternative to standard affinity chromatography. Methods for site-specific proteolysis of the fusion proteins to release native target proteins are also discussed.
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| 45. |
- Gulich, S., et al.
(författare)
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Stability towards alkaline conditions can be engineered into a protein ligand
- 2000
-
Ingår i: Journal of Biotechnology. - 0168-1656 .- 1873-4863. ; 80:2, s. 169-178
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Tidskriftsartikel (refereegranskat)abstract
- One of the problems with a proteinaceous affinity ligand is their sensitivity to alkaline conditions. Here, we show that a simple and straightforward strategy consisting of replacing all asparagine residues with other amino acids can dramatically improve the chemical stability of a protein towards alkaline conditions. As a model, a Streptococcal albumin-binding domain (ABD) was used. The engineered variant showed higher stability towards 0.5 M NaOH, as well as higher thermal stability compared to its native counterpart. This protein engineering approach could potentially also be used for other protein ligands to eliminate the sensitivity to alkaline cleaning-in-place (CIP) conditions.
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| 46. |
- Gummesson, Anders, 1973, et al.
(författare)
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Longitudinal plasma protein profiling of newly diagnosed type 2 diabetes
- 2021
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Ingår i: EBioMedicine. - : Elsevier B.V.. - 2352-3964. ; 63
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Tidskriftsartikel (refereegranskat)abstract
- Background: Comprehensive proteomics profiling may offer new insights into the dysregulated metabolic milieu of type 2 diabetes, and in the future, serve as a useful tool for personalized medicine. This calls for a better understanding of circulating protein patterns at the early stage of type 2 diabetes as well as the dynamics of protein patterns during changes in metabolic status. Methods: To elucidate the systemic alterations in early-stage diabetes and to investigate the effects on the proteome during metabolic improvement, we measured 974 circulating proteins in 52 newly diagnosed, treatment-naïve type 2 diabetes subjects at baseline and after 1 and 3 months of guideline-based diabetes treatment, while comparing their protein profiles to that of 94 subjects without diabetes. Findings: Early stage type 2 diabetes was associated with distinct protein patterns, reflecting key metabolic syndrome features including insulin resistance, adiposity, hyperglycemia and liver steatosis. The protein profiles at baseline were attenuated during guideline-based diabetes treatment and several plasma proteins associated with metformin medication independently of metabolic variables, such as circulating EPCAM. Interpretation: The results advance our knowledge about the biochemical manifestations of type 2 diabetes and suggest that comprehensive protein profiling may serve as a useful tool for metabolic phenotyping and for elucidating the biological effects of diabetes treatments. Funding: This work was supported by the Swedish Heart and Lung Foundation, the Swedish Research Council, the Erling Persson Foundation, the Knut and Alice Wallenberg Foundation, and the Swedish state under the agreement between the Swedish government and the county councils (ALF-agreement).
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| 47. |
- Gyllensten, Ulf B., et al.
(författare)
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Next Generation Plasma Proteomics Identifies High-Precision Biomarker Candidates for Ovarian Cancer
- 2022
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Ingår i: Cancers. - : MDPI AG. - 2072-6694. ; 14:7
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Tidskriftsartikel (refereegranskat)abstract
- Simple Summary Ovarian cancer is the eighth most common cancer among women and has a 5-year survival of only 30-50%. The survival is close to 90% for patients in stage I but only 20% for patients in stage IV. The presently available biomarkers have insufficient sensitivity and specificity for early detection and there is an urgent need to identify novel biomarkers. The aim of our study was to broadly measure protein biomarkers to find tests for the early detection of ovarian cancer. We found that combinations of 4-7 protein biomarkers can provide highly accurate detection of early- and late-stage ovarian cancer compared to benign conditions. The performance of the tests was then validated in a second independent cohort. Background: Ovarian cancer is the eighth most common cancer among women and has a 5-year survival of only 30-50%. The survival is close to 90% for patients in stage I but only 20% for patients in stage IV. The presently available biomarkers have insufficient sensitivity and specificity for early detection and there is an urgent need to identify novel biomarkers. Methods: We employed the Explore PEA technology for high-precision analysis of 1463 plasma proteins and conducted a discovery and replication study using two clinical cohorts of previously untreated patients with benign or malignant ovarian tumours (N = 111 and N = 37). Results: The discovery analysis identified 32 proteins that had significantly higher levels in malignant cases as compared to benign diagnoses, and for 28 of these, the association was replicated in the second cohort. Multivariate modelling identified three highly accurate models based on 4 to 7 proteins each for separating benign tumours from early-stage and/or late-stage ovarian cancers, all with AUCs above 0.96 in the replication cohort. We also developed a model for separating the early-stage from the late-stage achieving an AUC of 0.81 in the replication cohort. These models were based on eleven proteins in total (ALPP, CXCL8, DPY30, IL6, IL12, KRT19, PAEP, TSPAN1, SIGLEC5, VTCN1, and WFDC2), notably without MUCIN-16. The majority of the associated proteins have been connected to ovarian cancer but not identified as potential biomarkers. Conclusions: The results show the ability of using high-precision proteomics for the identification of novel plasma protein biomarker candidates for the early detection of ovarian cancer.
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| 48. |
- Hong, Mun-Gwan, et al.
(författare)
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Profiles of histidine-rich glycoprotein associate with age and risk of all-cause mortality
- 2020
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Ingår i: Life Science Alliance. - : Life Science Alliance, LLC. - 2575-1077. ; 3:10, s. e202000817-
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Tidskriftsartikel (refereegranskat)abstract
- Despite recognizing aging as a common risk factor of many human diseases, little is known about its molecular traits. To identify age-associated proteins circulating in human blood, we screened 156 individuals aged 50–92 using exploratory and multiplexed affinity proteomics assays. Profiling eight additional study sets (N = 3,987), performing antibody validation, and conducting a meta-analysis revealed a consistent age association (P = 6.61 × 10−6) for circulating histidine-rich glycoprotein (HRG). Sequence variants of HRG influenced how the protein was recognized in the immunoassays. Indeed, only the HRG profiles affected by rs9898 were associated with age and predicted the risk of mortality (HR = 1.25 per SD; 95% CI = 1.12–1.39; P = 6.45 × 10−5) during a follow-up period of 8.5 yr after blood sampling (IQR = 7.7–9.3 yr). Our affinity proteomics analysis found associations between the particular molecular traits of circulating HRG with age and all-cause mortality. The distinct profiles of this multipurpose protein could serve as an accessible and informative indicator of the physiological processes related to biological aging.
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| 49. |
- Hultin, Sara, et al.
(författare)
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AmotL2 links VE-cadherin to contractile actin fibres necessary for aortic lumen expansion
- 2014
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 5
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Tidskriftsartikel (refereegranskat)abstract
- The assembly of individual endothelial cells into multicellular tubes is a complex morphogenetic event in vascular development. Extracellular matrix cues and cell-cell junctional communication are fundamental to tube formation. Together they determine the shape of endothelial cells and the tubular structures that they ultimately form. Little is known regarding how mechanical signals are transmitted between cells to control cell shape changes during morphogenesis. Here we provide evidence that the scaffold protein amotL2 is needed for aortic vessel lumen expansion. Using gene inactivation strategies in zebrafish, mouse and endothelial cell culture systems, we show that amotL2 associates to the VE-cadherin adhesion complex where it couples adherens junctions to contractile actin fibres. Inactivation of amotL2 dissociates VE-cadherin from cytoskeletal tensile forces that affect endothelial cell shape. We propose that the VE-cadherin/amotL2 complex is responsible for transmitting mechanical force between endothelial cells for the coordination of cellular morphogenesis consistent with aortic lumen expansion and function.
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| 50. |
- Ibarra, Cristian, et al.
(författare)
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BCG-induced cytokine release in bladder cancer cells is regulated by Ca2+ signaling
- 2019
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Ingår i: Molecular Oncology. - : WILEY. - 1574-7891 .- 1878-0261. ; 13:2, s. 202-211
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Tidskriftsartikel (refereegranskat)abstract
- Bacillus Calmette-Guerin (BCG) is widely used in the clinic to effectively treat superficial urinary bladder cancer. However, a significant proportion of patients who fail to respond to BCG risk cystectomy or death. Though more than 3 million cancer treatments with BCG occur annually, surprisingly little is known about the initial signaling cascades activated by BCG. Here, we report that BCG induces a rapid intracellular Ca2+ (calcium ion) signal in bladder cancer cells that is essential for activating the transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappa B) and for synthesizing and secreting proinflammatory cytokines, including interleukin 8 (IL-8). A similar Ca2+ response was observed when cells were exposed to the supernatant of BCG. Studying cellular molecular mechanisms involved in the BCG signaling event, we found pivotal roles for phospholipase C and the Toll-like receptor 4. Further assessment revealed that this signaling pathway induces synthesis of IL-8, whereas exocytosis appeared to be controlled by global Ca2+ signaling. These results shed new light on the molecular mechanisms underlying BCG treatment of bladder cancer, which can help in improving therapeutic efficacy and reducing adverse side effects.
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