Project description:Emerging evidence indicates that metabolic enzymes perform moonlighting functions during tumor progression, including the modulation of chemoresistance. However, the underlying mechanisms of these functions remain elusive. In this study, utilizing a metabolic CRISPR-Cas9 knockout library screen, we observed that loss of Glutamate-cysteine ligase modifier subunit (GCLM), a rate-limiting enzyme in glutathione biosynthesis, noticeably heightens the sensitivity of colorectal cancer (CRC) cells to platinum-based chemotherapy. Mechanistically, we unveil a noncanonical mechanism through which nuclear GCLM competitively interacts with NF-kappa-B-repressing factor (NKRF), a known inhibitor of NF-κB signaling, to promote NF-κB activity and subsequently facilitate chemoresistance. In response to platinum drug treatment, P38 MAPK phosphorylates GCLM at T17, resulting in its recognition by importin a5 and subsequent nuclear translocation. Furthermore, elevated expression of nuclear GCLM correlates with unfavorable prognosis and poor benefit from standard chemotherapy. Overall, our work shed light on the essential nonmetabolic role and posttranslational regulatory mechanism of GCLM in enhancing NF-κB activity and subsequent chemoresistance.
Project description:Non-alcoholic steatohepatitis (NASH) and type 2 diabetes are closely linked, yet the pathophysiological mechanisms underpinning this bidirectional relationship remain unresolved. Using proteomic approaches, we interrogated hepatocyte protein secretion in two models of murine NASH to reveal striking hepatokine remodelling that is associated with insulin resistance and maladaptive lipid metabolism. We identify arylsulfatase A (ARSA) as a novel hepatokine that is upregulated in NASH and type 2 diabetes. This submission contains proteomic analysis of quadracep lipid rafts with or without overexpression of ARSA.
Project description:Platelets are involved in relevant physiological and pathophysiological processes such as hemostasis and tumor progression. These anucleate blood particles exert their functions essentially by releasing effector molecules such as proteins from intracellular granules and by generating and secreting specific lipids. In order to contribute to a systematic proteomics and eicosadomics analysis of platelets activated by calcium mobilization, we have investigated proteins and eicosanoids in lysates and secretomes of platelets activated with ionomycin. To this end, self-established shotgun proteomics and shotgun lipidomics platforms were used. Applying an FDR of less than 0.01 at both, peptide and protein level, a total of 2543 protein groups was identified. Several regulatory effects concerning proteins were determined, including protein secretion, degradation, as well as protein synthesis. Incorporation of 35S-methionine in platelet proteins determined by 2D-PAGE confirmed de-novo protein synthesis. Moreover, we were able to determine a complex set of eicosanoids synthesized by activated platelets, including 15S-HETE, prostaglandin E2 and pro-survival factors such as 12-HETE, as well as eicosanoids previously undescribed to have platelet functions, such as 9-HETE and 11-HETE. Proteomics data complemented the findings from lipidomics experiments and supported the generation of a map indicating pathways leading to eicosanoid production in platelets upon calcium mobilization.
Project description:Non-alcoholic steatohepatitis (NASH) and type 2 diabetes are closely linked, yet the pathophysiological mechanisms underpinning this bidirectional relationship remain unresolved. Using proteomic approaches, we interrogated hepatocyte protein secretion in two models of murine NASH to reveal striking hepatokine remodelling that is associated with insulin resistance and maladaptive lipid metabolism.
Project description:Breast cancer (BC) is an important disease with high incidence as well as mortality among women, and critical socio-economic impacts. In the past 50 years, it has become a major health problem for women worldwide with over 2 million new cases diagnosed in 2018. This represents about 12% of all new cancer cases, 25% of all cancers in women and more than 600.000 cases of deaths worldwide in 2018 (Bray et al, 2018). If the cancer is located only in the breast, the 5-year relative survival rate of people with BC is 99% but this rate decreases if it is spread to lymph nodes (85%) and more dramatically if diagnosed with distant metastasis (26%) (Howlader et al, 2019; Siegel et al, 2017). Metastatic process, or the spread of tumor cells throughout the body, is responsible for about 90% of cancer patient deaths (Chaffer et al, 2011) and represents the central clinical challenge of solid tumor oncology. The development and progression of BC are complex processes that involve hormonal factors as well as numerous genetic and epigenetic alterations. During the past 10 years, many studies have focused on the role of the tumor microenvironment and the peritumoral stromal fraction, composed of adipose tissue, cancer-associated fibroblasts, endothelial cells and immune cells as macrophages and leukocytes. During tumor progression, cancer cells will deeply modify their microenvironment which in return will promote the growth and dissemination of the tumor (Allen et al, 2011; Polanska et al, 2013). Adipose tissue, consisting of mainly mature adipocytes and progenitors (preadipocytes and adipose-derived stem cells (ADSCs), is the most abundant component surrounding BC cells. Adipose tissue exerts a major endocrine and secretory role, and represents then an essential actor in the inflammatory, angiogenic or remodeling responses of the extracellular matrix, which influences tumor behavior.
Project description:The possibility to combine untargeted proteomic workflows to more classical experimental approaches is continuously opening new insights in all branches of biological sciences. Deoxynivalenol (vomitoxin, DON) is a secondary metabolite produced by Fusarium spp. fungi and it is one of the most recurrent mycotoxins worldwide. DON is known to inhibit protein synthesis and as such to interact with the different cell types in multiple and complex ways. For the purpose of this study epidermoid squamous cell carcinoma cells A431 and primary human HEKn cells were incubated with DON for 24h and toxin dependent alteration of the proteome profile was observed in the nuclear and cytoplasmic fraction. In A431 cells, DON significantly down-regulated ribosomal proteins, as well as mitochondrial respiratory chain elements (OXPHOS regulation) and transport proteins (TOMM22; TOMM40; TOMM70A). In line with the impairment of the mitochondrial function, altered metabolic capability was observed, with particular target of the lipid synthesis machinery. Effect of the mycotoxin on cell membrane was verified by confocal microscopy (morphology) and by membrane fluidity measures (biophysical properties). The toxicological relevance of these findings was independently confirmed with the primary human keratinocytes HEKn.
Project description:While the most important players of inflammation have been well described, a systematic analysis of the proteins fulfilling the effector functionalities during inflammation has not yet been undertaken. Here we present a systematic proteome study of inflammatory activated primary human endothelial cells and fibroblasts. Cells were stimulated with interleukin 1-beta and fractionated in order to obtain secreted, cytoplasmic and nuclear protein fractions. Proteins were submitted to a data-dependent bottom up analytical platform using a QExactive orbitrap and the MaxQuant software for protein identification and label-free quantification. Results were further combined with similarly generated data previously obtained from the analysis of inflammatory activated peripheral blood mononuclear cells. Applying an FDR of less than 0.01 at both peptide and protein level, a total of 8235 protein groups assembled from 163858 peptides was identified. Comparative proteome analysis allowed us to determine proteins regulated in each kind of cells during inflammation. Remarkably, cells were working on similar inflammation-related tasks, however, by regulating different proteins. Thus, we were able to determine cell type-specific inflammatory signatures, apparently resulting from cell type-specific regulatory mechanisms. Hallmarks of inflammation emerged from these findings, representing commonly and cell type-specific responsibilities of cells during inflammation.
Project description:While the most important players of inflammation have been well described, a systematic analysis of the proteins fulfilling the effector functionalities during inflammation has not yet been undertaken. Here we present a systematic proteome study of inflammatory activated primary human endothelial cells and fibroblasts. Cells were stimulated with interleukin 1-beta and fractionated in order to obtain secreted, cytoplasmic and nuclear protein fractions. Proteins were submitted to a data-dependent bottom up analytical platform using a QExactive orbitrap and the MaxQuant software for protein identification and label-free quantification. Results were further combined with similarly generated data previously obtained from the analysis of inflammatory activated peripheral blood mononuclear cells. Applying an FDR of less than 0.01 at both peptide and protein level, a total of 8235 protein groups assembled from 163858 peptides was identified. Comparative proteome analysis allowed us to determine proteins regulated in each kind of cells during inflammation. Remarkably, cells were working on similar inflammation-related tasks, however, by regulating different proteins. Thus, we were able to determine cell type-specific inflammatory signatures, apparently resulting from cell type-specific regulatory mechanisms. Hallmarks of inflammation emerged from these findings, representing commonly and cell type-specific responsibilities of cells during inflammation.
Project description:Fibroblasts have only recently been identified as important effector cells in inflammation. In this study, human dermal fibroblasts were inflammatory stimulated with interleukin-1beta and comprehensively analysed with respect to proteins, eicosanoids and metabolites. For eicosadomics, we have established a data-dependent shotgun analysis method capable of identifying inflammation-regulated lipids of yet unknown function. Several classical inflammatory agonists were found induced, including PGA2, PGB2, PGE2 and TXB2, but also modulators such as PGA3 and PGE3, while 8-HETE and several HODE family members remained unaffected. Using targeted metabolomics, several acylcarnithins, phosphatitylcholins and sphingomyelins were found significantly downregulated. Proteome profiling with orbitrap-MS demonstrated the strong induction of several chemokines, metalloproteinases and other effector molecules. Treatment of stimulated fibroblasts with dexamethasone almost completely abrogated the formation of all inflammation-induced eicosanoids and restored levels of acylcarnithins back to normal. As expected, the secretion of IL-6, MMP1, MMP3, CXCL2 and CXCL3 was strongly down-regulated. However, instead of counter-regulating, dexamethasone further enhanced consequences of inflammatory stimulation with respect to CXCL1, CXCL6, complement C3 as well as sphingomyelins. Shotgun secretome data were confirmed by targeted analysis with triple-quadrupol-MS. These molecules have been described to be involved in chronic inflammation. In peripheral blood mononuclear cells, actually dexamethasone successfully downregulated the formation of all detectable inflammation mediators. The present data suggest that successful pharmacological abrogation of the formation of lipid inflammatory mediators in fibroblasts may not suffice to suppress the release of several other powerful inflammatory mediators which we thus understand to be capable of establishing chronic inflammation states.
Project description:Fibroblasts have only recently been identified as important effector cells in inflammation. In this study, human dermal fibroblasts were inflammatory stimulated with interleukin-1beta and comprehensively analysed with respect to proteins, eicosanoids and metabolites. For eicosadomics, we have established a data-dependent shotgun analysis method capable of identifying inflammation-regulated lipids of yet unknown function. Several classical inflammatory agonists were found induced, including PGA2, PGB2, PGE2 and TXB2, but also modulators such as PGA3 and PGE3, while 8-HETE and several HODE family members remained unaffected. Using targeted metabolomics, several acylcarnithins, phosphatitylcholins and sphingomyelins were found significantly downregulated. Proteome profiling with orbitrap-MS demonstrated the strong induction of several chemokines, metalloproteinases and other effector molecules. Treatment of stimulated fibroblasts with dexamethasone almost completely abrogated the formation of all inflammation-induced eicosanoids and restored levels of acylcarnithins back to normal. As expected, the secretion of IL-6, MMP1, MMP3, CXCL2 and CXCL3 was strongly down-regulated. However, instead of counter-regulating, dexamethasone further enhanced consequences of inflammatory stimulation with respect to CXCL1, CXCL6, complement C3 as well as sphingomyelins. Shotgun secretome data were confirmed by targeted analysis with triple-quadrupol-MS. These molecules have been described to be involved in chronic inflammation. In peripheral blood mononuclear cells, actually dexamethasone successfully downregulated the formation of all detectable inflammation mediators. The present data suggest that successful pharmacological abrogation of the formation of lipid inflammatory mediators in fibroblasts may not suffice to suppress the release of several other powerful inflammatory mediators which we thus understand to be capable of establishing chronic inflammation states.