phosphorylation profiling
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Numerous studies have reported that increased interleukin 6 (IL-6) and soluble IL-6 receptor (sIL-6) levels induce inflammatory conditions. However, the exact mechanisms by which IL-6 drives inflammatory conditions remain unclear. Therefore, we investigated the potential role of IL-6/sIL-6R in inducing energy metabolism, including glycolysis, oxidative phosphorylation, lactate secretion and Akt/mTOR phosphorylation, in Jurkat cells, and whether IL-6 would increase the risk of developing inflammatory conditions due to the high metabolic profile of the T cells. Jurkat CD4 T-cell lines were stimulated with IL-6/sIL-6R for 24 h prior to 48-h stimulation with anti-CD3/CD28. Lactate secretion, glycolysis and oxidative phosphorylation levels were characterized using the Seahorse XF analyser. The Akt and mTOR phosphorylation status was detected using Western blotting. IL-6/sIL-6R significantly induced glycolysis and oxidative phosphorylation and their related parameters, including glycolytic capacity and maximal respiration, followed by significantly increased lactate secretion. Akt and mTOR phosphorylation were increased, which could have resulted from energy metabolism. Here we show that IL-6 enhanced the metabolic profile of Jurkat cells. This effect could have consequences for the metabolism-related signalling pathways, including Akt and mTOR, suggesting that IL-6 might promote T-cell energy metabolism, where T-cell hyperactivity might increase the inflammatory disease risk. The findings should be validated using studies on primary cells isolated from humans.
- MeSH
- energetický metabolismus * účinky léků MeSH
- fosforylace účinky léků MeSH
- glykolýza účinky léků MeSH
- interleukin-6 * metabolismus MeSH
- Jurkat buňky MeSH
- kyselina mléčná metabolismus MeSH
- lidé MeSH
- oxidativní fosforylace účinky léků MeSH
- protoonkogenní proteiny c-akt * metabolismus MeSH
- signální transdukce * účinky léků MeSH
- TOR serin-threoninkinasy * metabolismus MeSH
- zánět * metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Cardiovascular diseases are associated with an altered cardiomyocyte metabolism. Because of a shortage of human heart tissue, experimental studies mostly rely on alternative approaches including animal and cell culture models. Since the use of isolated primary cardiomyocytes is limited, immortalized cardiomyocyte cell lines may represent a useful tool as they closely mimic human cardiomyocytes. This study is focused on the AC16 cell line generated from adult human ventricular cardiomyocytes. Despite an increasing number of studies employing AC16 cells, a comprehensive proteomic, bioenergetic, and oxygen-sensing characterization of proliferating vs. differentiated cells is still lacking. Here, we provide a comparison of these two stages, particularly emphasizing cell metabolism, mitochondrial function, and hypoxic signaling. Label-free quantitative mass spectrometry revealed a decrease in autophagy and cytoplasmic translation in differentiated AC16, confirming their phenotype. Cell differentiation led to global increase in mitochondrial proteins [e.g. oxidative phosphorylation (OXPHOS) proteins, TFAM, VWA8] reflected by elevated mitochondrial respiration. Fatty acid oxidation proteins were increased in differentiated cells, whereas the expression levels of proteins associated with fatty acid synthesis were unchanged and glycolytic proteins were decreased. There was a profound difference between proliferating and differentiated cells in their response to hypoxia and anoxia-reoxygenation. We conclude that AC16 differentiation leads to proteomic and metabolic shifts and altered cell response to oxygen deprivation. This underscores the requirement for proper selection of the particular differentiation state during experimental planning.NEW & NOTEWORTHY Proliferating and differentiated AC16 cell lines exhibit distinct proteomic and metabolic profiles with critical implications for experimental design. Proliferating cells predominantly utilize glycolysis and are highly sensitive to hypoxia, whereas differentiated cells display enhanced mitochondrial biogenesis, oxidative phosphorylation, and resistance to anoxia-reoxygenation. These findings provide novel insights into the metabolic adaptations during differentiation and highlight the necessity of selecting the appropriate cellular stage to ensure accurate experimental outcomes.
- MeSH
- buněčná diferenciace * fyziologie MeSH
- buněčné linie MeSH
- energetický metabolismus MeSH
- hypoxie buňky fyziologie MeSH
- kardiomyocyty * metabolismus MeSH
- lidé MeSH
- mitochondriální proteiny metabolismus MeSH
- mitochondrie * metabolismus MeSH
- oxidativní fosforylace MeSH
- proliferace buněk MeSH
- proteomika metody MeSH
- signální transdukce * fyziologie MeSH
- srdeční mitochondrie * metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Extramedullary disease (EMD) is a high-risk feature of multiple myeloma (MM) and remains a poor prognostic factor, even in the era of novel immunotherapies. Here, we applied spatial transcriptomics (RNA tomography for spatially resolved transcriptomics [tomo-seq] [n = 2] and 10x Visium [n = 12]) and single-cell RNA sequencing (n = 3) to a set of 14 EMD biopsies to dissect the 3-dimensional architecture of tumor cells and their microenvironment. Overall, infiltrating immune and stromal cells showed both intrapatient and interpatient variations, with no uniform distribution over the lesion. We observed substantial heterogeneity at the copy number level within plasma cells, including the emergence of new subclones in circumscribed areas of the tumor, which is consistent with genomic instability. We further identified the spatial expression differences between GPRC5D and TNFRSF17, 2 important antigens for bispecific antibody therapy. EMD masses were infiltrated by various immune cells, including T cells. Notably, exhausted TIM3+/PD-1+ T cells diffusely colocalized with MM cells, whereas functional and activated CD8+ T cells showed a focal infiltration pattern along with M1 macrophages in tumor-free regions. This segregation of fit and exhausted T cells was resolved in the case of response to T-cell-engaging bispecific antibodies. MM and microenvironment cells were embedded in a complex network that influenced immune activation and angiogenesis, and oxidative phosphorylation represented the major metabolic program within EMD lesions. In summary, spatial transcriptomics has revealed a multicellular ecosystem in EMD with checkpoint inhibition and dual targeting as potential new therapeutic avenues.
OBJECTIVES: Our previous studies have demonstrated that the Damage Associated Molecular Pattern (DAMP) protein, S100A4, is overexpressed in the involved skin and peripheral blood of patients with SSc. It is associated with skin and lung involvement, and disease activity. By contrast, lack of S100A4 prevented the development of experimental dermal fibrosis. Herein we aimed to evaluate the effect of murine anti-S100A4 mAb 6B12 in the treatment of preestablished experimental dermal fibrosis. METHODS: The effects of 6B12 were assessed at therapeutic dosages in a modified bleomycin-induced dermal fibrosis mouse model by evaluating fibrotic (dermal thickness, proliferation of myofibroblasts, hydroxyproline content, phosphorylated Smad3-positive cell count) and inflammatory (leukocytes infiltrating the lesional skin, systemic levels of selected cytokines and chemokines) outcomes, and transcriptional profiling (RNA sequencing). RESULTS: Treatment with 7.5 mg/kg 6B12 attenuated and might even reduce pre-existing dermal fibrosis induced by bleomycin as evidenced by reduction in dermal thickness, myofibroblast count and collagen content. These antifibrotic effects were mediated by the downregulation of TGF-β/Smad signalling and partially by reducing the number of leukocytes infiltrating the lesional skin and decrease in the systemic levels of IL-1α, eotaxin, CCL2 and CCL5. Moreover, transcriptional profiling demonstrated that 7.5 mg/kg 6B12 also modulated several profibrotic and proinflammatory processes relevant to the pathogenesis of SSc. CONCLUSION: Targeting S100A4 by the 6B12 mAb demonstrated potent antifibrotic and anti-inflammatory effects on bleomycin-induced dermal fibrosis and provided further evidence for the vital role of S100A4 in the pathophysiology of SSc.
- MeSH
- alarminy * MeSH
- bleomycin toxicita MeSH
- fibróza MeSH
- kůže * patologie MeSH
- lidé MeSH
- modely nemocí na zvířatech MeSH
- monoklonální protilátky farmakologie MeSH
- myši MeSH
- S100 kalcium vázající protein A4 genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
OBJECTIVE: S100A4 is a DAMP protein. S100A4 is overexpressed in patients with systemic sclerosis (SSc), and levels correlate with organ involvement and disease activity. S100A4-/- mice are protected from fibrosis. The aim of this study was to assess the antifibrotic effects of anti-S100A4 monoclonal antibody (mAb) in murine models of SSc and in precision cut skin slices of patients with SSc. METHODS: The effects of anti-S100A4 mAbs were evaluated in a bleomycin-induced skin fibrosis model and in Tsk-1 mice with a therapeutic dosing regimen. In addition, the effects of anti-S100A4 mAbs on precision cut SSc skin slices were analyzed by RNA sequencing. RESULTS: Inhibition of S100A4 was effective in the treatment of pre-established bleomycin-induced skin fibrosis and in regression of pre-established fibrosis with reduced dermal thickening, myofibroblast counts, and collagen accumulation. Transcriptional profiling demonstrated targeting of multiple profibrotic and proinflammatory processes relevant to the pathogenesis of SSc on targeted S100A4 inhibition in a bleomycin-induced skin fibrosis model. Moreover, targeted S100A4 inhibition also modulated inflammation- and fibrosis-relevant gene sets in precision cut SSc skin slices in an ex vivo trial approach. Selected downstream targets of S100A4, such as AMP-activated protein kinase, calsequestrin-1, and phosphorylated STAT3, were validated on the protein level, and STAT3 inhibition was shown to prevent the profibrotic effects of S100A4 on fibroblasts in human skin. CONCLUSION: Inhibition of S100A4 confers dual targeting of inflammatory and fibrotic pathways in complementary mouse models of fibrosis and in SSc skin. These effects support the further development of anti-S100A4 mAbs as disease-modifying targeted therapies for SSc.
- MeSH
- bleomycin * MeSH
- fibróza * MeSH
- kůže * patologie účinky léků metabolismus MeSH
- lidé MeSH
- modely nemocí na zvířatech * MeSH
- monoklonální protilátky * farmakologie terapeutické užití MeSH
- myši MeSH
- S100 kalcium vázající protein A4 * genetika metabolismus MeSH
- systémová sklerodermie * farmakoterapie genetika MeSH
- transkripční faktor STAT3 metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
TP53 gene abnormalities represent the most important biomarker in chronic lymphocytic leukemia (CLL). Altered protein modifications could also influence p53 function, even in the wild-type protein. We assessed the impact of p53 protein phosphorylations on p53 functions as an alternative inactivation mechanism. We studied p53 phospho-profiles induced by DNA-damaging agents (fludarabine, doxorubicin) in 71 TP53-intact primary CLL samples. Doxorubicin induced two distinct phospho-profiles: profile I (heavily phosphorylated) and profile II (hypophosphorylated). Profile II samples were less capable of activating p53 target genes upon doxorubicin exposure, resembling TP53-mutant samples at the transcriptomic level, whereas standard p53 signaling was triggered in profile I. ATM locus defects were more common in profile II. The samples also differed in the basal activity of the hypoxia pathway: the highest level was detected in TP53-mutant samples, followed by profile II and profile I. Our study suggests that wild-type TP53 CLL cells with less phosphorylated p53 show TP53-mutant-like behavior after DNA damage. p53 hypophosphorylation and the related lower ability to respond to DNA damage are linked to ATM locus defects and the higher basal activity of the hypoxia pathway.
- MeSH
- ATM protein genetika metabolismus MeSH
- chronická lymfatická leukemie * genetika MeSH
- doxorubicin farmakologie MeSH
- fosforylace MeSH
- geny p53 MeSH
- hypoxie genetika MeSH
- lidé MeSH
- nádorový supresorový protein p53 * metabolismus MeSH
- poškození DNA MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Dilated cardiomyopathy (DCM) is a major cause of heart failure and carries a high mortality rate. Myocardial recovery in DCM-related heart failure patients is highly variable, with some patients having little or no response to standard drug therapy. A genome-wide association study may agnostically identify biomarkers and provide novel insight into the biology of myocardial recovery in DCM. METHODS: A genome-wide association study for change in left ventricular ejection fraction was performed in 686 White subjects with recent-onset DCM who received standard pharmacotherapy. Genome-wide association study signals were subsequently functionally validated and studied in relevant cellular models to understand molecular mechanisms that may have contributed to the change in left ventricular ejection fraction. RESULTS: The genome-wide association study identified a highly suggestive locus that mapped to the 5'-flanking region of the CDCP1 (CUB [complement C1r/C1s, Uegf, and Bmp1] domain containing protein 1) gene (rs6773435; P=7.12×10-7). The variant allele was associated with improved cardiac function and decreased CDCP1 transcription. CDCP1 expression was significantly upregulated in human cardiac fibroblasts (HCFs) in response to the PDGF (platelet-derived growth factor) signaling, and knockdown of CDCP1 significantly repressed HCF proliferation and decreased AKT (protein kinase B) phosphorylation. Transcriptomic profiling after CDCP1 knockdown in HCFs supported the conclusion that CDCP1 regulates HCF proliferation and mitosis. In addition, CDCP1 knockdown in HCFs resulted in significantly decreased expression of soluble ST2 (suppression of tumorigenicity-2), a prognostic biomarker for heart failure and inductor of cardiac fibrosis. CONCLUSIONS: CDCP1 may play an important role in myocardial recovery in recent-onset DCM and mediates its effect primarily by attenuating cardiac fibrosis.
- MeSH
- antigeny nádorové terapeutické užití MeSH
- celogenomová asociační studie MeSH
- dilatační kardiomyopatie * metabolismus MeSH
- fibróza MeSH
- funkce levé komory srdeční MeSH
- lidé MeSH
- molekuly buněčné adheze metabolismus MeSH
- srdeční selhání * MeSH
- tepový objem MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Small molecule Bruton's tyrosine kinase (BTK) inhibitors have been developed for the treatment of various haemato-oncological diseases, and ibrutinib was approved as the first BTK inhibitor for anticancer therapy in 2013. Previous reports proved the receptor kinase human epidermal growth factor receptor 2 (HER2) to be a valid off-target kinase of ibrutinib and potentially other irreversible BTK inhibitors, as it possesses a druggable cysteine residue in the active site of the enzyme. These findings suggest ibrutinib as a candidate drug for repositioning in HER2-positive breast cancer (BCa). This subtype of breast cancer belongs to one of the most common classes of breast tumours, and its prognosis is characterized by a high rate of recurrence and tumour invasiveness. Based on their similar kinase selectivity profiles, we investigated the anticancer effect of zanubrutinib, evobrutinib, tirabrutinib and acalabrutinib in different BCa cell lines and sought to determine whether it is linked with targeting the epidermal growth factor receptor family (ERBB) pathway. We found that zanubrutinib is a potential inhibitor of the HER2 signalling pathway, displaying an antiproliferative effect in HER2-positive BCa cell lines. Zanubrutinib effectively inhibits the phosphorylation of proteins in the ERBB signalling cascade, including the downstream kinases Akt and ERK, which mediate key signals ensuring the survival and proliferation of cancer cells. We thus propose zanubrutinib as another suitable candidate for repurposing in HER2-amplified solid tumours.
Microtubule-associated protein 2 (MAP2) is an important neuronal target of extracellular signal-regulated kinase 2 (ERK2) involved in Raf signaling pathways, but mechanistic details of MAP2 phosphorylation are unclear. Here, we used NMR spectroscopy to quantitatively describe the kinetics of phosphorylation of individual serines and threonines in the embryonic MAP2 variant MAP2c. We carried out real-time monitoring of phosphorylation to discover major phosphorylation sites that were not identified in previous studies relying on specific antibodies. Our comparison with the phosphorylation of MAP2c by a model cyclin-dependent kinase CDK2 and with phosphorylation of the MAP2c homolog Tau revealed differences in phosphorylation profiles that explain specificity of regulation of biological functions of MAP2c and Tau. To probe the molecular basis of the regulatory effect of ERK2, we investigated the interactions of phosphorylated and unphosphorylated MAP2c by NMR with single-residue resolution. As ERK2 phosphorylates mostly outside the regions binding microtubules, we studied the binding of proteins other than tubulin, namely regulatory subunit RIIα of cAMP-dependent PKA, adapter protein Grb2, Src homology domain 3 of tyrosine kinases Fyn and Abl, and ERK2 itself. We found ERK2 phosphorylation interfered mostly with binding to proline-rich regions of MAP2c. Furthermore, our NMR experiments in SH-SY5Y neuroblastoma cell lysates showed that the kinetics of dephosphorylation are compatible with in-cell NMR studies and that residues targeted by ERK2 and PKA are efficiently phosphorylated in the cell lysates. Taken together, our results provide a deeper characterization of MAP2c phosphorylation and its effects on interactions with other proteins.
- MeSH
- extracelulárním signálem regulované MAP kinasy * metabolismus MeSH
- fosforylace MeSH
- lidé MeSH
- mikrotubuly metabolismus MeSH
- nádorové buněčné linie MeSH
- proteinkinasy řízené prolinem * metabolismus MeSH
- proteiny asociované s mikrotubuly * metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The human transient receptor potential canonical 5 (TRPC5) is a calcium-permeable, nonselective cation channel expressed in the central and peripheral nervous system and also in other tissues such as the kidney, synovium, and odontoblasts. TRPC5 has been recently confirmed to play a key role in spontaneous, inflammatory mechanical, and cold pain. Although TRPC5 activation is known to be cold sensitive, it is unclear whether this property is intrinsic to the channel protein and whether or to what extent it may be determined by the cellular environment. In this study, we explored the cold sensitivity of human TRPC5 at the single-channel level using transiently transfected HEK293T cells. Upon decreasing the temperature, the channel demonstrated prolonged mean open dwell times and a robust increase in the open probability (Po ), whereas the amplitude of unitary currents decreased ~1.5-fold per 10°C of temperature difference. In the absence of any agonists, the temperature dependence of Po was sigmoidal, with a steep slope within the temperature range of 16°C-11°C, and exhibited saturation below 8-5°C. Thermodynamic analysis revealed significant changes in enthalpy and entropy, suggesting that substantial conformational changes accompany cold-induced gating. The mutant channel T970A, in which the regulation downstream of G-protein coupled receptor signaling was abrogated, exhibited higher basal activity at room temperature and a less steep temperature response profile, with an apparent threshold below 22°C. An even more pronounced decrease in the activation threshold was observed in a mutant that disrupted the electrostatic interaction of TRPC5 with the endoplasmic reticulum calcium sensor stromal interaction molecule 1. Thus, TRPC5 exhibits features of an intrinsically cold-gated channel; its sensitivity to cold tightly depends on the phosphorylation status of the protein and intracellular calcium homeostasis.
- MeSH
- buněčná membrána metabolismus MeSH
- HEK293 buňky MeSH
- kationtové kanály TRPC * genetika metabolismus MeSH
- lidé MeSH
- vápník * metabolismus MeSH
- vápníkové kanály metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
