| 1. |
- Beusch, Christian M., et al.
(författare)
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Analysis of local extracellular matrix identifies different aetiologies behind bicuspid and tricuspid aortic valve degeneration and suggests therapies
- 2023
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Ingår i: Cellular and Molecular Life Sciences (CMLS). - : Springer Nature. - 1420-682X .- 1420-9071. ; 80:9
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Tidskriftsartikel (refereegranskat)abstract
- Aortic valve degeneration (AVD) is a life-threatening condition that has no medical treatment and lacks individual therapies. Although extensively studied with standard approaches, aetiologies behind AVD are unclear. We compared abundances of extracellular matrix (ECM) proteins from excised valve tissues of 88 patients with isolated AVD of normal tricuspid (TAV) and congenital bicuspid aortic valves (BAV), quantified more than 1400 proteins per ECM sample by mass spectrometry, and demonstrated that local ECM preserves molecular cues of the pathophysiological processes. The BAV ECM showed enrichment with fibrosis markers, namely Tenascin C, Osteoprotegerin, and Thrombospondin-2. The abnormal physical stress on BAV may cause a mechanical injury leading to a continuous Tenascin C-driven presence of myofibroblasts and persistent fibrosis. The TAV ECM exhibited enrichment with Annexin A3 (p = 1.1 x 10(-16) and the fold change 6.5) and a significant deficit in proteins involved in high-density lipid metabolism. These results were validated by orthogonal methods. The difference in the ECM landscape suggests distinct aetiologies between AVD of BAV and TAV; warrants different treatments of the patients with BAV and TAV; elucidates the molecular basis of AVD; and implies possible new therapeutic approaches. Our publicly available database (human_avd_ecm.surgsci. uu.se) is a rich source for medical doctors and researchers who are interested in AVD or heart ECM in general. Systematic proteomic analysis of local ECM using the methods described here may facilitate future studies of various tissues and organs in development and disease.
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| 2. |
- Gubat, Johannes, 1986-, et al.
(författare)
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Comprehensive Target Screening and Cellular Profiling of the Cancer-Active Compound b-AP15 Indicate Abrogation of Protein Homeostasis and Organelle Dysfunction as the Primary Mechanism of Action
- 2022
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Ingår i: Frontiers in Oncology. - : Frontiers Media SA. - 2234-943X. ; 12
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Tidskriftsartikel (refereegranskat)abstract
- Dienone compounds have been demonstrated to display tumor-selective anti-cancer activity independently of the mutational status of TP53. Previous studies have shown that cell death elicited by this class of compounds is associated with inhibition of the ubiquitin-proteasome system (UPS). Here we extend previous findings by showing that the dienone compound b-AP15 inhibits proteasomal degradation of long-lived proteins. We show that exposure to b-AP15 results in increased association of the chaperones VCP/p97/Cdc48 and BAG6 with proteasomes. Comparisons between the gene expression profile generated by b-AP15 to those elicited by siRNA showed that knock-down of the proteasome-associated deubiquitinase (DUB) USP14 is the closest related to drug response. USP14 is a validated target for b-AP15 and we show that b-AP15 binds covalently to two cysteines, Cys203 and Cys257, in the ubiquitin-binding pocket of the enzyme. Consistent with this, deletion of USP14 resulted in decreased sensitivity to b-AP15. Targeting of USP14 was, however, found to not fully account for the observed proteasome inhibition. In search for additional targets, we utilized genome-wide CRISPR/Cas9 library screening and Proteome Integral Solubility Alteration (PISA) to identify mechanistically essential genes and b-AP15 interacting proteins respectively. Deletion of genes encoding mitochondrial proteins decreased the sensitivity to b-AP15, suggesting that mitochondrial dysfunction is coupled to cell death induced by b-AP15. Enzymes known to be involved in Phase II detoxification such as aldo-ketoreductases and glutathione-S-transferases were identified as b-AP15-targets using PISA. The finding that different exploratory approaches yielded different results may be explained in terms of a “target” not necessarily connected to the “mechanism of action” thus highlighting the importance of a holistic approach in the identification of drug targets. We conclude that b-AP15, and likely also other dienone compounds of the same class, affect protein degradation and proteasome function at more than one level.
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| 3. |
- Guédron, Stéphane, et al.
(författare)
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Holocene variations in Lake Titicaca water level and their implications for sociopolitical developments in the central Andes
- 2023
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 120:2
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Tidskriftsartikel (refereegranskat)abstract
- Holocene climate in the high tropical Andes was characterized by both gradual and abrupt changes, which disrupted the hydrological cycle and impacted landscapes and societies. High-resolution paleoenvironmental records are essential to contextualize archaeological data and to evaluate the sociopolitical response of ancient societies to environmental variability. Middle-to-Late Holocene water levels in Lake Titicaca were reevaluated through a transfer function model based on measurements of organic carbon stable isotopes, combined with high-resolution profiles of other geochemical variables and paleoshoreline indicators. Our reconstruction indicates that following a prolonged low stand during the Middle Holocene (4000 to 2400 BCE), lake level rose rapidly ~15 m by 1800 BCE, and then increased another 3 to 6 m in a series of steps, attaining the highest values after ~1600 CE. The largest lake-level increases coincided with major sociopolitical changes reported by archaeologists. In particular, at the end of the Formative Period (500 CE), a major lake-level rise inundated large shoreline areas and forced populations to migrate to higher elevation, likely contributing to the emergence of the Tiwanaku culture.
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| 4. |
- Kaldmäe, Margit, et al.
(författare)
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A “spindle and thread” mechanism unblocks p53 translation by modulating N-terminal disorder
- 2022
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Ingår i: Structure. - : Elsevier BV. - 0969-2126 .- 1878-4186. ; 30:5, s. 733-742, e1-e7
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Tidskriftsartikel (refereegranskat)abstract
- Disordered proteins pose a major challenge to structural biology. A prominent example is the tumor suppressor p53, whose low expression levels and poor conformational stability hamper the development of cancer therapeutics. All these characteristics make it a prime example of “life on the edge of solubility.” Here, we investigate whether these features can be modulated by fusing the protein to a highly soluble spider silk domain (NT∗). The chimeric protein displays highly efficient translation and is fully active in human cancer cells. Biophysical characterization reveals a compact conformation, with the disordered transactivation domain of p53 wrapped around the NT∗ domain. We conclude that interactions with NT∗ help to unblock translation of the proline-rich disordered region of p53. Expression of partially disordered cancer targets is similarly enhanced by NT∗. In summary, we demonstrate that inducing co-translational folding via a molecular “spindle and thread” mechanism unblocks protein translation in vitro.
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| 5. |
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| 6. |
- Sabatier, Pierre, et al.
(författare)
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An integrative proteomics method identifies a regulator of translation during stem cell maintenance and differentiation
- 2021
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Ingår i: Nature Communications. - : Springer Nature. - 2041-1723. ; 12
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Tidskriftsartikel (refereegranskat)abstract
- To characterize molecular changes during cell type transitions, the authors develop a method to simultaneously measure protein expression and thermal stability changes. They apply this approach to study differences between human pluripotent stem cells, their progenies, parental and allogeneic cells. Detailed characterization of cell type transitions is essential for cell biology in general and particularly for the development of stem cell-based therapies in regenerative medicine. To systematically study such transitions, we introduce a method that simultaneously measures protein expression and thermal stability changes in cells and provide the web-based visualization tool ProteoTracker. We apply our method to study differences between human pluripotent stem cells and several cell types including their parental cell line and differentiated progeny. We detect alterations of protein properties in numerous cellular pathways and components including ribosome biogenesis and demonstrate that modulation of ribosome maturation through SBDS protein can be helpful for manipulating cell stemness in vitro. Using our integrative proteomics approach and the web-based tool, we uncover a molecular basis for the uncoupling of robust transcription from parsimonious translation in stem cells and propose a method for maintaining pluripotency in vitro.
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| 7. |
- Sabatier, Pierre
(författare)
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Plurifaceted proteomics in studying cellular dynamics and action mechanisms of anticancer drugs
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Mass spectrometry (MS)-based proteomics has developed tremendously in recent years and was the leading technology for many novel methods to study protein chemistry. Contrary to classical approaches based on Western blot, MS-based approaches are mostly unbiased. In addition to protein expression levels, today several protein chemical properties can be examined by MS-based proteomics making it unique in comparison to transcriptomics approaches. These properties include post-translational modifications (PTMs), protein localization, synthesis/degradation, and lastly protein thermal stability, adding novel dimensions or facets to characterize the proteome. However, these facets are difficult to combine as they are mainly orthogonal and are therefore often analyzed separately. This thesis presents a simplified and higher throughput version of current protein stability analyses and showcases the advantages of combined/merged analysis of proteomics facets including our new method, as well as expression and redox proteomics to study anticancer treatments and cellular dynamics. In paper I, we studied the dynamics of cancer cells in vitro with and without anticancer treatment over the course of 48 h by monitoring protein expression every 6 h. We discovered that naturally occurring proteome variations are on par with anticancer treatment killing 50% of the cells after 48 h. Then, we acquired a deep proteomics dataset of untreated HCT116 and A375 cell lines. Surprisingly, we observed downregulation of proteins involved in cell division and upregulation of proteins involved in metabolism as early as 12 h after treatment, suggesting that growth inhibition happens earlier than usually assumed and even at low cell confluence. In paper II we developed the proteome integral solubility alteration (PISA) assay that increases the throughput of pre-existing drug target deconvolution methods based on protein stability/solubility measurements and reduces the analysis time and cost as well as sample requirements. We provided theoretical calculations showing that the integral of the curve correlates well with melting temperature estimations in Thermal Proteome Profiling (TPP) and tested our assumptions with publicly available TPP datasets. Then we performed a proof of principle experiment using the well-studied methotrexate (MTX) and 5-fluorouracile (5-FU) as test drugs highlighting the targets as outliers with our method. Furthermore, we demonstrated that PISA assay can also be used for concentration series analysis as in 2D-TPP. Finally, we showcased the higher throughput of PISA compared to TPP by simultaneously analyzing nine drugs in one multiplexed analysis. In paper III we used a combination of chemical proteomics approaches to study the target and mechanism of action (MOA) of Auranofin (AF) (Ridaura®). Functional Identification of Target by Expression Proteomics (FITExP), TPP, and redox proteomics combined highlighted that thioredoxin reductase 1 (TXNRD1) is indeed a top target hit of AF, and that the main MOA of the drug is through disruption of the redox balance in the cell. Finally, we showed that protein thermal shifts can be associated with altered cysteine oxidation levels in proteins, suggesting that TPP is suitable to study disulfide bond formation/reduction and map some cysteines to the active sites of sulfiredoxin 1 (SRXN1) and peroxiredoxin 5 (PRDX5) as examples. Overall, our study demonstrates that using only one of the proteomics methods is not sufficient to accurately pinpoint drug target and MOA, but a combination of multiple complementary methods should be used instead. Following the success of our strategy in paper III, we decided to utilize the same strategy in paper IV using PISA developed in paper II instead of TPP, to study two novel inhibitors of TXNRD1, namely TRi-1 and TRi-2. For these studies, we used the mouse cancer cell lines B16-F10 and Lewis lung carcinoma (LLC) to provide a comprehensive analysis of the therapeutic effects of the inhibitors. Since we showed that Txnrd1 (the mouse version of the human TXNRD1 protein) is a main target of AF, we decided to use it as our reference point to evaluate the specificity of the two new compounds. AF had a broader effect on the proteome than TRi-1 and TRi-2 and had more target hits in PISA analyses. This suggested that AF has lower specificity than TRi-1 and TRi-2. TRi-1 was the most specific Txnrd1 inhibitor with a better target ranking in FITExP and the least target hits in PISA followed by TRi-2 and AF. Thus, we showed that TRi-1 and TRi-2 are indeed more specific inhibitors of Txnrd1. In addition to these findings, we also highlighted that only AF triggers a high nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidant response, suggesting that this response is not necessarily Txnrd1-dependent. Finally, we detected selenocysteine-specific elongation factor (Eefsec), mini-chromosome maintenance complex-binding protein (Mcmbp), glycogen synthase kinase-3 (Gsk3) a and b, as target hits of AF, which would explain at least partially three different effects of AF treatment. Collectively, our data represents a resource for redox biologists interested in Txnrd1 inhibition. Our approach provides a framework for target deconvolution using proteomics approaches. Finally, in paper V we studied the dynamics of the proteome in transition between various cell types. We reprogrammed human foreskin fibroblasts (hFF) into induced pluripotent stem cells (iPSCs), which we differentiated through embryoid bodies (EBs) formation. We examined protein expression and stability after each cell type transition using PISA-Express, a new version of the PISA assay developed in paper II. We merged the readout from protein expression and thermal stability in one analysis using Sankey diagrams to detect changes in protein properties during proteome transitions resulting in the ProteoTracker web interface (http://www.proteotracker.genexplain.com/). Using this innovative analysis, we discovered that ribosomes are less stable in pluripotent stem cells (PSCs) compared to differentiated cells and that this difference stems from the deficiency of one ribosome maturation factor, Shwachman-Bodian-Diamond syndrome protein (SBDS). Knock-down (KD) of SBDS slowed down translation and increased expression of the master pluripotency markers homeobox protein NANOG (NANOG), and octamer-binding transcription factor 4 (OCT4). Collectively, we developed a new method for simultaneous analysis of protein thermal stability and expression, a new analysis and visualization tool, and provided evidence that control of translation through ribosome biogenesis is a natural mechanism used by PSCs to maintain the pluripotency state.
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| 8. |
- Saei, Amir Ata, et al.
(författare)
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Comprehensive chemical proteomics for target deconvolution of the redox active drug auranofin
- 2020
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Ingår i: Redox Biology. - : Elsevier BV. - 2213-2317. ; 32
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Tidskriftsartikel (refereegranskat)abstract
- Chemical proteomics encompasses novel drug target deconvolution methods in which compound modification is not required. Herein we use Thermal Proteome Profiling, Functional Identification of Target by Expression Proteomics and multiplexed redox proteomics for deconvolution of auranofin targets to aid elucidation of its mechanisms of action. Auranofin (Ridaura (R)) was approved for treatment of rheumatoid arthritis in 1985. Because several clinical trials are currently ongoing to repurpose auranofin for cancer therapy, comprehensive characterization of its targets and effects in cancer cells is important. Together, our chemical proteomics tools confirmed thioredoxin reductase 1 (TXNRD1, EC:1.8.1.9) as a main auranofin target, with perturbation of oxidoreductase pathways as the top mechanism of drug action. Additional indirect targets included NFKB2 and CHORDC1. Our comprehensive data can be used as a proteomic signature resource for further analyses of the effects of auranofin. Here we also assessed the orthogonality and complementarity of different chemical proteomics methods that can furnish invaluable mechanistic information and thus the approach can facilitate drug discovery efforts in general.
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| 9. |
- Saei, Amir Ata, et al.
(författare)
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System-wide identification and prioritization of enzyme substrates by thermal analysis
- 2021
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Ingår i: Nature Communications. - : Springer Nature. - 2041-1723. ; 12:1
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Tidskriftsartikel (refereegranskat)abstract
- Despite the immense importance of enzyme-substrate reactions, there is a lack of general and unbiased tools for identifying and prioritizing substrate proteins that are modified by the enzyme on the structural level. Here we describe a high-throughput unbiased proteomics method called System-wide Identification and prioritization of Enzyme Substrates by Thermal Analysis (SIESTA). The approach assumes that the enzymatic post-translational modification of substrate proteins is likely to change their thermal stability. In our proof-of-concept studies, SIESTA successfully identifies several known and novel substrate candidates for selenoprotein thioredoxin reductase 1, protein kinase B (AKT1) and poly-(ADP-ribose) polymerase-10 systems. Wider application of SIESTA can enhance our understanding of the role of enzymes in homeostasis and disease, opening opportunities to investigate the effect of post-translational modifications on signal transduction and facilitate drug discovery.
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| 10. |
- Urbonaviciute, Vilma, et al.
(författare)
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Therapy targeting antigen-specific T cells by a peptide-based tolerizing vaccine against autoimmune arthritis
- 2023
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - Stockholm : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 120:25
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Tidskriftsartikel (refereegranskat)abstract
- A longstanding goal has been to find an antigen-specific preventive therapy, i.e., a vaccine, for autoimmune diseases. It has been difficult to find safe ways to steer the targeting of natural regulatory antigen. Here, we show that the administration of exog-enous mouse major histocompatibility complex class II protein bounding a unique galactosylated collagen type II (COL2) peptide (Aq-galCOL2) directly interacts with the antigen-specific TCR through a positively charged tag. This leads to expanding a VISTA-positive nonconventional regulatory T cells, resulting in a potent dominant suppressive effect and protection against arthritis in mice. The therapeutic effect is dom-inant and tissue specific as the suppression can be transferred with regulatory T cells, which downregulate various autoimmune arthritis models including antibody-induced arthritis. Thus, the tolerogenic approach described here may be a promising dominant antigen-specific therapy for rheumatoid arthritis, and in principle, for autoimmune diseases in general.
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