| 1. |
- Arias, Carolina, et al.
(författare)
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Nuclear proteome analysis of Chlamydomonas with response to CO2 limitation
- 2020
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Ingår i: Algal Research. - : Elsevier. - 2211-9264. ; 46
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Tidskriftsartikel (refereegranskat)abstract
- Chlamydomonas reinhardtii is a unicellular green alga that can survive at a wide range of inorganic carbon (Ci) concentrations by regulating the activity of a CO2-concentrating mechanism (CCM) as well as other cellular functions. Under CO2 limited conditions, C. reinhardtii cells display a wide range of adaptive responses including changes in photosynthetic electron transport, mitochondria localization in the cells, the structure of the pyrenoid starch sheath, and primary metabolism. In addition to these functional and structural changes, gene and protein expression are also affected. Several physiological aspects of the CO2 response mechanism have been studied in detail. However, the regulatory components (transcription factors and transcriptional regulators) involved in this process are not fully characterized. Here we report a comprehensive analysis of the C. reinhardtii nuclear proteome using liquid chromatography electrospray ionization spectrometry (LC-ESI-MS). The study aims to identify the proteins that govern adaptation to varying CO2 concentrations in Chlamydomonas. The nuclear proteome of C. reinhardtii cells grown in the air at high (5%) and low (0.04%) CO2 concentrations were analyzed. Using this approach, we identified 1378 proteins in total, including 90 putative transcription factors and 27 transcriptional regulators. Characterization of these new regulatory components could shed light on the molecular mechanisms underlying acclimation to CO2 stress.
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| 2. |
- Dutta, Tanmoy, 1998, et al.
(författare)
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Mitochondrial amidoxime-reducing component 1 p.Ala165Thr increases protein degradation mediated by the proteasome.
- 2024
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Ingår i: Liver international : official journal of the International Association for the Study of the Liver. - 1478-3231.
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Tidskriftsartikel (refereegranskat)abstract
- Metabolic dysfunction-associated steatotic liver disease (MASLD) is a global health concern with no effective and specific drug treatment available. The rs2642438 minor allele in mitochondrial amidoxime-reducing component 1 (MARC1) results in an aminoacidic substitution (p.Ala165Thr) and associates with protection against MASLD. However, the mechanisms behind this protective effect are unknown. In this study, we examined the consequences of this aminoacidic substitution on protein stability and subcellular localization.We overexpressed the human MARC1 A165 (wild-type) or 165T (mutant) in vivo in mice and in vitro in human hepatoma cells (HepG2 and HuH-7), generated several mutants at position 165 by in situ mutagenesis and then examined protein levels. We also generated HepG2 cells stably overexpressing MARC1 A165 or 165T to test the effect of this substitution on MARC1 subcellular localization.MARC1 165T overexpression resulted in lower protein levels than A165 both in vivo and in vitro. Similarly, any mutant at position 165 showed lower protein levels compared to the wild-type protein. We showed that the 165T mutant protein is polyubiquitinated and its degradation is accelerated through lysine-48 ubiquitin-mediated proteasomal degradation. We also showed that the 165T substitution does not affect the MARC1 subcellular localization.This study shows that alanine at position 165 in MARC1 is crucial for protein stability, and the threonine substitution at this position leads to a hypomorphic protein variant due to lower protein levels. Our result supports the notion that lowering hepatic MARC1 protein level may be a successful therapeutic strategy for treating MASLD.
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| 3. |
- Iglesias, Maria Jesus, et al.
(författare)
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Combined Chromatin and Expression Analysis Reveals Specific Regulatory Mechanisms within Cytokine Genes in the Macrophage Early Immune Response
- 2012
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 7:2, s. e32306-
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Tidskriftsartikel (refereegranskat)abstract
- Macrophages play a critical role in innate immunity, and the expression of early response genes orchestrate much of the initial response of the immune system. Macrophages undergo extensive transcriptional reprogramming in response to inflammatory stimuli such as Lipopolysaccharide (LPS). To identify gene transcription regulation patterns involved in early innate immune responses, we used two genome-wide approaches - gene expression profiling and chromatin immunoprecipitation-sequencing (ChIP-seq) analysis. We examined the effect of 2 hrs LPS stimulation on early gene expression and its relation to chromatin remodeling (H3 acetylation; H3Ac) and promoter binding of Sp1 and RNA polymerase II phosphorylated at serine 5 (S5P RNAPII), which is a marker for transcriptional initiation. Our results indicate novel and alternative gene regulatory mechanisms for certain proinflammatory genes. We identified two groups of upregulated inflammatory genes with respect to chromatin modification and promoter features. One group, including highly up-regulated genes such as tumor necrosis factor (TNF), was characterized by H3Ac, high CpG content and lack of TATA boxes. The second group, containing inflammatory mediators (interleukins and CCL chemokines), was up-regulated upon LPS stimulation despite lacking H3Ac in their annotated promoters, which were low in CpG content but did contain TATA boxes. Genome-wide analysis showed that few H3Ac peaks were unique to either +/-LPS condition. However, within these, an unpacking/expansion of already existing H3Ac peaks was observed upon LPS stimulation. In contrast, a significant proportion of S5P RNAPII peaks (approx 40%) was unique to either condition. Furthermore, data indicated a large portion of previously unannotated TSSs, particularly in LPS-stimulated macrophages, where only 28% of unique S5P RNAPII peaks overlap annotated promoters. The regulation of the inflammatory response appears to occur in a very specific manner at the chromatin level for specific genes and this study highlights the level of fine-tuning that occurs in the immune response.
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| 4. |
- Lehtiö, Janne, et al.
(författare)
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Proteogenomics of non-small cell lung cancer reveals molecular subtypes associated with specific therapeutic targets and immune-evasion mechanisms
- 2021
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Ingår i: Nature Cancer. - : Springer Science and Business Media LLC. - 2662-1347. ; 2:11, s. 1224-1242
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Tidskriftsartikel (refereegranskat)abstract
- Despite major advancements in lung cancer treatment, long-term survival is still rare and a deeper understanding of molecular phenotypes would allow the identification of specific cancer dependencies and immune-evasion mechanisms. Here we performed in-depth mass-spectrometry-based proteogenomic analysis of 141 tumors representing all major histologies of non-small cell lung cancer (NSCLC). We identified six distinct proteome subtypes with striking differences in immune cell composition and subtype-specific expression of immune checkpoints. Unexpectedly, high neoantigen burden was linked to global hypomethylation and complex neoantigens mapped to genomic regions, such as endogenous retroviral elements and introns, in immune-cold subtypes. Further, we linked immune evasion with LAG-3 via STK11 mutation-dependent HNF1A activation and FGL1 expression. Finally, we develop a data-independent acquisition mass-spectrometry-based NSCLC subtype classification method, validate it in an independent cohort of 208 NSCLC cases and demonstrate its clinical utility by analyzing an additional cohort of 84 late-stage NSCLC biopsy samples.
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| 5. |
- Pecori, Riccardo, et al.
(författare)
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ADAR1-mediated RNA editing promotes B cell lymphomagenesis
- 2023
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Ingår i: iScience. - : Cell Press. - 2589-0042. ; 26:6
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Tidskriftsartikel (refereegranskat)abstract
- Diffuse large B cell lymphoma (DLBCL) is one of the most common types of aggressive lymphoid malignancies. Here, we explore the contribution of RNA editing to DLBCL pathogenesis. We observed that DNA mutations and RNA editing events are often mutually exclusive, suggesting that tumors can modulate pathway outcomes by altering sequences at either the genomic or the transcriptomic level. RNA editing targets transcripts within known disease-driving pathways such as apoptosis, p53 and NF-kappa B signaling, as well as the RIG-I-like pathway. In this context, we show that ADAR1-mediated editing within MAVS transcript positively correlates with MAVS protein expression levels, associating with increased interferon/NF-kappa B signaling and T cell exhaustion. Finally, using targeted RNA base editing tools to restore editing within MAVS 3 ' UTR in ADAR1-deficient cells, we demonstrate that editing is likely to be causal to an increase in downstream signaling in the absence of activation by canonical nucleic acid receptor sensing.
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| 6. |
- Pirmoradian, Mohammad
(författare)
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Proteomics of neurodegenerative diseases using novel online isoelectric point separation
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The work presented in this thesis describes an instrument, developed for separation of proteins and peptides based on corresponding isoelectric point (pI) values, to empower mass spectrometry-based proteomics analysis. The main objectives are the instrument development and optimization, as well as clinical applications in biomarker discovery, particularly for neurodegenerative disorders. The thesis is based on five scientific papers that focus on three main stages; (i) development and optimization of the device for separation of proteins and peptides by pI that are well integrated with tandem mass spectrometry (ii) optimization of the device for intact blood plasma protein fractionation for application in biomarker discovery, and (iii) biomarker discovery in blood plasma for early diagnosis of Alzheimer disease. Within the first part of the work, a novel multiple-junction capillary isoelectric focusing fractionator (MJ-CIEF) is developed (Paper I). Subsequently, the resolving power and reproducibility of fractionation are improved, and in addition, a novel algorithm is developed to calculate the identified peptides’ pI (Paper II). Moreover, to achieve the aim of deep proteomics, a multi-parameter optimization of the LC-MS/MS pipeline is performed (Paper III). In the second part, an online desalinator is developed and coupled to the device, for direct buffer-exchange and isoelectric separation of intact human blood plasma/serum proteins. The developed pipeline achieves the increased depth of the proteome analysis and provides additional information on the pI of identified proteins, as another dimension of information in biomarker discovery by proteomics (Paper IV). The last part of the thesis is focused on the application of the developed method in biomarker discovery for early diagnosis of Alzheimer disease. A panel of new potential biomarkers is introduced based on the abundance changes, as well as shifts in the pI values. By means of the pI information, the protein concentration in a narrow pH range around 7.4 reveals increased levels in patients with progressive Alzheimer disease compared to stable ones. Proteome analysis of this particular pI region also suggests several potential proteins as biomarkers for early diagnosis of the disease (Paper V). Taken together, this thesis demonstrates emerging applications of peptides and proteins fractionation by pI in deep proteomics. The development of MJ-CIEF facilitates online separation of peptides and proteins from small amounts of samples in a fast format, automatable, cost-effective and compatible with mass spectrometry analysis. Further biomarker discovery in the narrow range of pH around 7.4 of blood proteome is suggested for early diagnosis of neurodegenerative diseases.
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| 7. |
- Zhang, Bo, et al.
(författare)
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Covariation of Peptide Abundances Accurately Reflects Protein Concentration Differences
- 2017
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Ingår i: Molecular & Cellular Proteomics. - : American Society for Biochemistry and Molecular Biology. - 1535-9476 .- 1535-9484. ; 16:5, s. 936-948
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Tidskriftsartikel (refereegranskat)abstract
- Most implementations of mass spectrometry-based proteomics involve enzymatic digestion of proteins, expanding the analysis to multiple proteolytic peptides for each protein. Currently, there is no consensus of how to summarize peptides' abundances to protein concentrations, and such efforts are complicated by the fact that error control normally is applied to the identification process, and do not directly control errors linking peptide abundance measures to protein concentration. Peptides resulting from suboptimal digestion or being partially modified are not representative of the protein concentration. Without a mechanism to remove such unrepresentative peptides, their abundance adversely impacts the estimation of their protein's concentration. Here, we present a relative quantification approach, Diffacto, that applies factor analysis to extract the covariation of peptides' abundances. The method enables a weighted geometrical average summarization and automatic elimination of incoherent peptides. We demonstrate, based on a set of controlled label-free experiments using standard mixtures of proteins, that the covariation structure extracted by the factor analysis accurately reflects protein concentrations. In the 1% peptide-spectrum match-level FDR data set, as many as 11% of the peptides have abundance differences incoherent with the other peptides attributed to the same protein. If not controlled, such contradicting peptide abundance have a severe impact on protein quantifications. When adding the quantities of each protein's three most abundant peptides, we note as many as 14% of the proteins being estimated as having a negative correlation with their actual concentration differences between samples. Diffacto reduced the amount of such obviously incorrectly quantified proteins to 1.6%. Furthermore, by analyzing clinical data sets from two breast cancer studies, our method revealed the persistent proteomic signatures linked to three subtypes of breast cancer. We conclude that Diffacto can facilitate the interpretation and enhance the utility of most types of proteomics data.
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