The complexity of omes - the key cellular ensembles (genome and epigenome, transcriptome, proteome, and metabolome) - is becoming increasingly understood in terms of big-data analysis, the omics. Amongst these, proteomics provides a global description of quantitative and qualitative alterations of protein expression (or protein abundance in body fluids) in response to physiologic or pathologic processes while metabolomics offers a functional portrait of the physiological state by quantifying metabolite abundances in biological samples. Here, we summarize how different techniques of proteomic and metabolic analysis can be used to define key biochemical characteristics of pheochromocytomas/paragangliomas (PPGL). The significance of omics in understanding features of PPGL biology that might translate to improved diagnosis and treatment will be highlighted.
BACKGROUND: Cystathionine β-synthase (CBS)-deficient homocystinuria (HCU) is an inherited disorder of sulfur amino acid metabolism with varying severity and organ complications, and a limited knowledge about underlying pathophysiological processes. Here we aimed at getting an in-depth insight into disease mechanisms using a transgenic mouse model of HCU (I278T). METHODS: We assessed metabolic, proteomic and sphingolipidomic changes, and mitochondrial function in tissues and body fluids of I278T mice and WT controls. Furthermore, we evaluated the efficacy of methionine-restricted diet (MRD) in I278T mice. RESULTS: In WT mice, we observed a distinct tissue/body fluid compartmentalization of metabolites with up to six-orders of magnitude differences in concentrations among various organs. The I278T mice exhibited the anticipated metabolic imbalance with signs of an increased production of hydrogen sulfide and disturbed persulfidation of free aminothiols. HCU resulted in a significant dysregulation of liver proteome affecting biological oxidations, conjugation of compounds, and metabolism of amino acids, vitamins, cofactors and lipids. Liver sphingolipidomics indicated upregulation of the pro-proliferative sphingosine-1-phosphate signaling pathway. Liver mitochondrial function of HCU mice did not seem to be impaired compared to controls. MRD in I278T mice improved metabolic balance in all tissues and substantially reduced dysregulation of liver proteome. CONCLUSION: The study highlights distinct tissue compartmentalization of sulfur-related metabolites in normal mice, extensive metabolome, proteome and sphingolipidome disruptions in I278T mice, and the efficacy of MRD to alleviate some of the HCU-related biochemical abnormalities.
- MeSH
- cystathionin-beta-synthasa * metabolismus nedostatek genetika MeSH
- homocystinurie * metabolismus genetika MeSH
- játra * metabolismus MeSH
- lipidomika metody MeSH
- metabolomika * metody MeSH
- mitochondrie metabolismus MeSH
- modely nemocí na zvířatech * MeSH
- myši transgenní * MeSH
- myši MeSH
- proteom metabolismus MeSH
- proteomika * metody MeSH
- sfingolipidy * metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
There is no biomarker reflecting right ventricular dysfunction in HFrEF patients used in clinical practice. We have aimed to look for a circulating marker of RV dysfunction employing a quantitative proteomic strategy. The Olink Proteomics Multiplex panels (Cardiovascular Disease II, III, Cardiometabolic, and Inflammation Target Panels) identified FGF-23 to be the most differentially abundant (more than 2.5-fold) in blood plasma of HF patients with severe RV dysfunction (n = 30) compared to those with preserved RV function (n = 31). A subsequent ELISA-based confirmatory analysis of circulating FGF-23 in a large cohort of patients (n = 344, 72.7% NYHA III/IV, LVEF 22.5%, 54.1% with moderate/severe RV dysfunction), followed by multivariable regression analysis, revealed that the plasma FGF-23 level was most significantly associated with RV dysfunction grade (p = 0.0004) and congestion in the systemic circulation (p = 0.03), but not with LV-ejection fraction (p = 0.69) or estimated glomerular filtration rate (eGFR, p = 0.08). FGF-23 was associated with the degree of RV dysfunction in both sub-cohorts (i.e. in patients with and without congestion, p < 0.0001). The association between FGF-23 and RV-dysfunction remained significant after the adjustment for BNP (p = 0.01). In contrast, when adjusted for BNP, FGF-23 was no longer associated with LV dysfunction (p = 0.59). The Cox proportional hazard model revealed that circulating FGF-23 was significantly associated with adverse outcomes even after adjusting for BNP, LVEF, RV dysfunction grade and eGFR. Circulating FGF-23 is thus a biomarker of right ventricular dysfunction in HFrEF patients regardless of congestion status.
BACKGROUND: Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors. New drug targets and proteins that would assist sensitive PPGL imagining could improve therapy and quality of life of patients with PPGL, namely those with recurrent or metastatic disease. Using a combined proteomic strategy, we looked for such clinically relevant targets among integral membrane proteins (IMPs) upregulated on the surface of tumor cells and non-membrane druggable enzymes in PPGL. METHODS: We conducted a detailed proteomic analysis of 22 well-characterized human PPGL samples and normal chromaffin tissue from adrenal medulla. A standard quantitative proteomic analysis of tumor lysate, which provides information largely on non-membrane proteins, was accompanied by specific membrane proteome-aimed methods, namely glycopeptide enrichment using lectin-affinity, glycopeptide capture by hydrazide chemistry, and enrichment of membrane-embedded hydrophobic transmembrane segments. RESULTS: The study identified 67 cell surface integral membrane proteins strongly upregulated in PPGL compared to control chromaffin tissue. We prioritized the proteins based on their already documented direct role in cancer cell growth or progression. Increased expression of the seven most promising drug targets (CD146, CD171, ANO1, CD39, ATP8A1, ACE and SLC7A1) were confirmed using specific antibodies. Our experimental strategy also provided expression data for soluble proteins. Among the druggable non-membrane enzymes upregulated in PPGL, we identified three potential drug targets (SHMT2, ARG2 and autotaxin) and verified their upregulated expression. CONCLUSIONS: Application of a combined proteomic strategy recently presented as "Pitchfork" enabled quantitative analysis of both, membrane and non-membrane proteome, and resulted in identification of 10 potential drug targets in human PPGL. Seven membrane proteins localized on the cell surface and three non-membrane druggable enzymes proteins were identified and verified as significantly upregulated in PPGL. All the proteins have been previously shown to be upregulated in several human cancers, and play direct role in cancer progression. Marked upregulation of these proteins along with their localization and established direct roles in tumor progression make these molecules promising candidates as drug targets or proteins for sensitive PPGL imaging.
- Publikační typ
- časopisecké články MeSH
Závěrečná zpráva o řešení grantu Agentury pro zdravotnický výzkum MZ ČR
nestr.
Pheochromocytoma and paraganglioma (PHEO/PGL) are rare neuroendocrine tumors arising from chromaffin cells of adrenal medulla and from extra-adrenal sympathetic and parasympathetic ganglia, respectively. The aim of the project is to characterize the molecular pathways (both, causal and associated) involved in PHEO/PGL development and progression in order to provide the basis for future sensitive diagnosis and knowledge-based targeted therapy of this rare disease. Top-notch proteomic analyses of patient tumor samples combined with transcriptome profiling using RNA sequencing technology will provide detailed and comprehensive quantitative proteome and transcriptome description of individual PHEO/PGL subtypes and will identify the underlying molecular processes and molecules exploitable as drug targets. Additionally, the proposed proteo-genomic “tour de force” will identify molecules that provide specific information about disease presence, type or progression – diagnostic and/or prognostic biomarkers of PHEO/PGL.
Feochormocytomy a pargangliomy (PHEO/PGL) jsou vzácné neuro-endokrinní nádory původem z chromafinních buněk dřeně nadledvin (PHEO) nebo extra-drenálních sympatických a parasympatických ganglií (PGL). Cílem projektu je budoucí zpřesnění diagnózy a zefektivnění terapie PHEO/PGL cestou podrobné charakterizace molekulárních procesů, drah a molekul spojených se vznikem a progresí PHEO/PGL. Detailní proteomická analýza (LC-MS/MS) pacientských vzorků PHEO/PGL kombinovaná s profilováním kompletního transkriptomu metodou sekvenace RNA (RNA-Seq) poskytne informaci o specifických procesech zodpovědných nebo asociovaných s tímto onemocněním. Tyto specifické molekuly a procesy jsou potenciálními zásahovými místy „druggable targets“ budoucích terapií a rovněž možnými diagnostickými či prognostickými markery.
- Klíčová slova
- feochromocytom, paragangliom, pheochromocytoma, paraganglioma, proteom, transkriptom, transcriptomics, proteomics, Hmotnostní spektrometrie, Mass Spectrometry,
- NLK Publikační typ
- závěrečné zprávy o řešení grantu AZV MZ ČR
Závěrečná zpráva o řešení grantu Agentury pro zdravotnický výzkum MZ ČR
nestr.
Advanced heart failure (HF) is associated with poor pronosis, but consumes the major portion of treatment resources. Advanced HF is often biventricular- there is right ventricular (RV) dysfunction accompanying left-ventricular (LV) dysfunction. Prediction of RV dysfunction onset in the context of LV dysfunction is difficult, but critically important for proper timing of further HF therapy (i.e. heart transplantation, left-ventricle asisst device implantation). The goal of the project is to identify proteins which are differentially expressed in myocardium of patients with biventricular failure compared to patients with LV dysfunction but preserved RV function and to control individuals without HF (organ donors). Current „top-notch“ proteomic approaches enable the identification of up to 10000 proteins, almost equaling the number of proteins typially expressed in a normal cell. Identification of these biomarker proteins in the circulation (plasma) can be a useful tool supporting clinical decision making. Identification of differentially expressed proteins can be useful not only for diagnotics or for prognostic assessment, but may identify also new therapeutic targets. A detailed proteomic analysis of pooled myocardial samples of patients undergoing heart transplantation (HTx, discovery cohort) and control samples will be performed. Samples of patients with normal RV function (upper quartile of the distribution of RV function) and RV dysfuction (lower quartile of the distribution of the RV function) will be compared with healthy controls (HF-free organ donor hearts not used for transplantnation). Differential expression of identified proteins will be verified in individual samples of the original and also another cohort of patients undergoing HTx (verification cohort). Verified and prioritized list of potential markers will be tested for detectability and altered concentration in plasma in patients from both cohorts. The ability of prioritized and verified candidate biomarkers to predict the presence of biventricular failure will be studied on the preexisting set of patients with advanced heart failure undergoing a thorough examination at IKEM; this cohort will be used to test the biomarkers for their prognostic power. The ability of biomarkers to predict RV dysfunction following LVAD implantation will be tested on another preexisting set of patients that underwent this intervention.
Pokročilé srdeční selhání je spojeno s velmi špatnou prognózou a zároveň spotřebovává největší část zdrojů pro léčbu tohoto onemocnění. Pokročilé srdeční selhání je velmi často biventrikulární – je přítomna dysfunkce nejen levé (LK), ale i pravé komory (PK). Predikce rozvoje dysfunkce pravé komory v kontextu levostranného selhání je obtížná, ale je kriticky důležitá pro správné načasování terapií jako jako transplantace srdce nebo implantace podpory levého srdce (LVAD). Cílem projektu je identifikovat bílkoviny diferenciálně exprimované v myokardu pacientů s biventrikulárním selháním oproti pacientům se srdečním selháním a zachovalou funkcí PK a kontrolám (orgánoví dárci). Současné proteomické techniky umožňují identifikaci až 10 000 proteinů, což téměř odpovídá počtu proteinů v běžné buňce. Detekce takových proteinů v krvi by byla užitečným nástrojem podporující klinické rozhodování. Identifikace diferenciálně exprimovaných proteinů může hrát roli nejen pro diagnostiku nebo pro zpřesnění odhadu prognózy, ale může také vést k identifikaci nových terapeutických cílů. Bude provedena detailní proteomická analýza směsných vzorků myokardu pacientů, kteří podstoupili transplantaci (Tx) srdce („discovery“ kohorta) a kontrol. Budou porovnány vzorky pacientů s normální funkcí PK (horní kvartil ditribuce funkce PK) a s dysfunkcí PK (dolní kvartil distribuce funkce PK). Diferenciální exprese identifikovaných proteinů bude následně verifikována v individuálních vzorcích myokardu původní i další kohorty Tx pacientů (verifikační kohorta). Verifikovaný a prioritizovaný seznam potenciálních markerů bude dále testován na přítomnost a změny koncentrace v séru pacientů obou kohort. Schopnost takto verifikovaných potenciálních biomarkerů predikovat přítomnost biventrikulárního selhání bude dále studována na preexistujícím setu pacientů s pokročilým srdečním selháním podstupujících podrobné vyšetření v IKEM, na této kohortě bude rovněž testována schopnost biomarkerů predikovat prognózu. Schopnost predikce výskytu selhání PK po implantaci LVAD bude sledována na dalším preexistujícím setu pacientů, kteří podstoupili tuto intervenci.
- Klíčová slova
- prognóza, biomarkery, prognosis, biomarkers, Heart failure, srdeční selhání, proteomics, proteomika, selhání pravé komory, right-ventricular failure,
- NLK Publikační typ
- závěrečné zprávy o řešení grantu AZV MZ ČR
Autotaxin, also known as ecto-nucleotide pyrophosphatase/phosphodiesterase family member 2, is a secreted glycoprotein that plays multiple roles in human physiology and cancer pathology. This protein, by converting lysophosphatidylcholine into lysophosphatidic acid, initiates a complex signalling cascade with significant biological implications. The article outlines the autotaxin gene and protein structure, expression regulation and physiological functions, but focuses mainly on the role of autotaxin in cancer development and progression. Autotaxin and lysophosphatidic acid signalling influence several aspects of cancer, including cell proliferation, migration, metastasis, therapy resistance, and interactions with the immune system. The potential of autotaxin as a diagnostic biomarker and promising drug target is also examined.
- MeSH
- fosfodiesterasy * genetika chemie metabolismus MeSH
- lidé MeSH
- lysofosfolipidy metabolismus MeSH
- nádory * MeSH
- signální transdukce MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
PURPOSE: The lack of reliable diagnostic and/or prognostic biomarkers for multiple sclerosis (MS) is the major obstacle to timely and accurate patient diagnosis in MS patients. To identify new proteins associated with MS we performed a detailed proteomic analysis of cerebrospinal fluid (CSF) of patients newly diagnosed with relapsing-remitting MS (RRMS) and healthy controls. MATERIAL: Reflecting significantly higher prevalence of MS in women we included only women patients and controls in the study. To eliminate a potential effect of therapy on the CSF composition, only the therapy-naïve patients were included. METHODS: Pooled CSF samples were processed in a technical duplicate, and labeled with stable-isotope coded TMT tags. To maximize the proteome coverage, peptide fractionation using 2D-LC preceded mass analysis using Orbitrap Fusion Tribrid Mass Spectrometer. Differential concentration of selected identified proteins between patients and controls was verified using specific antibodies. RESULTS: Of the identified 900 CSF proteins, we found 69 proteins to be differentially abundant between patients and controls. In addition to several proteins identified as differentially abundant in MS patients previously, we observed several linked to MS for the first time, namely eosinophil-derived neurotoxin and Nogo receptor. CONCLUSIONS: Our data confirm differential abundance of several previously proposed protein markers, and provide indirect support for involvement of copper-iron disbalance in MS. Most importantly, we identified two new differentially abundant CSF proteins that seem to be directly connected with myelin loss and axonal damage via TLR2 signaling and Nogo-receptor pathway in women newly diagnosed with RRMS.
- MeSH
- biologické markery mozkomíšní mok MeSH
- lidé MeSH
- mozkomíšní mok chemie metabolismus MeSH
- proteiny v mozkomíšním moku mozkomíšní mok MeSH
- proteom analýza metabolismus MeSH
- proteomika MeSH
- relabující-remitující roztroušená skleróza * diagnóza MeSH
- roztroušená skleróza * diagnóza MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
The number of people living with multiple sclerosis (MS) in developed countries is increasing. The management of patients is hindered by the absence of reliable laboratory tests accurately reflecting the disease activity. Extracellular vesicles (EVs) of different cell origin were reportedly elevated in MS patients. We assessed the diagnostic potential, with flow cytometry analysis, of fresh large EVs (lEVs), which scattered more light than the 590 nm silica beads and were isolated from the blood plasma of relapsing remitting MS patients. Venous blood was collected from 15 patients and 16 healthy controls (HC). The lEVs were isolated from fresh platelet-free plasma by centrifugation, labelled with antibodies and the presence of platelet (CD41+, CD36+), endothelial (CD105+), erythrocyte (CD235a+), leukocyte (CD45+, CD19+, CD3+) and phosphatidylserine (Annexin V+) positive lEVs was analyzed using standard flow cytometry. Cryo-electron microscopy was used to verify the presence of EVs in the analyzed plasma fractions. MS patients experiencing acute relapse had slightly reduced relative levels (% of positive lEVs) of CD105+, CD45+, CD3+, CD45+CD3+ or CD19+ labelled lEVs in comparison to healthy controls. An analysis of other markers or a comparison of absolute lEV counts (count of lEVs/μL) did not yield any significant differences. Our data do not support the hypothesis that the exacerbation of the disease in RRMS patients leads to an increased numbers of circulating plasma lEVs which can be monitored by standard flow cytometry.
- Publikační typ
- časopisecké články MeSH
Chronic myeloid leukemia (CML) is a malignant hematopoietic disorder distinguished by the presence of a BCR‑ABL1 fused oncogene with constitutive kinase activity. Targeted CML therapy by specific tyrosine kinase inhibitors (TKIs) leads to a marked improvement in the survival of the patients and their quality of life. However, the development of resistance to TKIs remains a critical issue for a subset of patients. The most common cause of resistance are numerous point mutations in the BCR‑ABL1 gene, followed by less common mutations and multiple mutation-independent mechanisms. Recently, exosomes, which are extracellular vesicles excreted from normal and tumor cells, have been associated with drug resistance and cancer progression. The aim of the present study was to characterize the exosomes released by imatinib‑resistant K562 (K562IR) cells. The K562IR‑derived exosomes were internalized by imatinib‑sensitive K562 cells, which thereby increased their survival in the presence of 2 µM imatinib. The exosomal cargo was subsequently analyzed to identify resistance‑associated markers using a deep label‑free quantification proteomic analysis. There were >3,000 exosomal proteins identified of which, 35 were found to be differentially expressed. From this, a total of 3, namely the membrane proteins, interferon‑induced transmembrane protein 3, CD146 and CD36, were markedly upregulated in the exosomes derived from the K562IR cells, and exhibited surface localization. The upregulation of these proteins was verified in the K562IR exosomes, and also in the K562IR cells. Using flow cytometric analysis, it was possible to further demonstrate the potential of CD146 as a cell surface marker associated with imatinib resistance in K562 cells. Taken together, these results suggested that exosomes and their respective candidate surface proteins could be potential diagnostic markers of TKI drug resistance in CML therapy.
- MeSH
- antigen CD146 metabolismus MeSH
- antigeny CD36 metabolismus MeSH
- apoptóza účinky léků MeSH
- bcr-abl fúzové proteiny antagonisté a inhibitory genetika MeSH
- buňky K562 MeSH
- chemorezistence MeSH
- chronická myeloidní leukemie farmakoterapie genetika patologie MeSH
- exozómy účinky léků metabolismus MeSH
- imatinib mesylát farmakologie terapeutické užití MeSH
- inhibitory proteinkinas farmakologie terapeutické užití MeSH
- lidé MeSH
- membránové proteiny metabolismus MeSH
- nádorové buněčné linie MeSH
- proteiny vázající RNA metabolismus MeSH
- viabilita buněk účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
