Project description:Increasing evidence suggests that in Amyotrophic Lateral Sclerosis (ALS) mutated RNA binding proteins acquire aberrant functions, leading to altered RNA metabolism with significant impact on encoded protein levels. Here, by taking advantage of a human induced Pluripotent Stem Cell (hiPSC)-based model, we aimed to gain insights on the impact of ALS mutant FUS on the motoneuron proteome. Label-free proteomics analysis by mass-spectrometry revealed upregulation of proteins involved in catabolic processes and oxidation-reduction, and downregulation of cytoskeletal proteins and factors directing neuron projection. Mechanistically, proteome alteration does not correlate with transcriptome changes. Rather, we observed a strong correlation with selective binding of mutant FUS to target mRNAs in their 3’UTR. Novel validated targets, selectively bound by mutant FUS only, include genes previously involved in familial or sporadic ALS, such as VCP, and regulators of membrane trafficking and cytoskeleton remodeling, such as ASAP1. These findings unveil a novel mechanism by which mutant FUS might intersect other pathogenic pathways in ALS patients’ motoneurons.
Project description:Many proteins undergo glycosylation in the endoplasmic reticulum (ER) and the Golgi apparatus. Altered glycosylation can manifest in serious, sometimes fatal malfunctions. We recently showed that mutations in the cytoplasmic protein GDP-mannose pyrophosphorylase A (GMPPA) cause a syndrome characterized by alacrima, achalasia, mental retardation and myopathic alterations. GMPPA acts as feedback inhibitor of GDP-mannose pyrophosphorylase B (GMPPB), which provides GDP-mannose as a substrate for protein glycosylation. Loss of GMPPA enhances incorporation of mannose into glycochains of various proteins, including α-dystroglycan (α-DG), a protein that links the extracellular matrix with the cytoskeleton. Here, we show that loss of GMPPA affects the functionality of the Golgi apparatus using different approaches. First, we show a fragmentation of the Golgi apparatus in skeletal muscle fibers and in neurons of GMPPA KO mice. A major reorganization is also evident by mass spectrometry of KO tissues with a regulation of several ER- and Golgi-resident proteins. We further show that loss of GMPPA increases the retention of α-DG in the ER. Notably, mannose supplementation can mimic changes in ER and Golgi structure and function in WT cells. In summary, our data underline the importance of a balanced mannose homeostasis for structure and function of the secretory pathway.
Project description:Mass spectrometry-based whole proteome analysis of parental and RFX7 knock-out U2OS cells treated with 10 µM Nutlin-3a or DMSO solvent control. Ten biological replicates were used.
Project description:Effective therapies for metastatic osteosarcoma (OS) remain a major clinical unmet need. Targeting mRNA translation in metastatic OS represents an attractive option, as selective translation under stress supports the rapid synthesis of cytoprotective proteins that facilitate metastatic competence. We therefore assessed eukaryotic translation factors in OS, revealing high expression of eIF4A1 in metastatic OS. The eIF4A1 inhibitor, CR-1-31B, potently inhibited metastatic OS growth in vitro and reduced lung tumor burden in orthotopic mouse models. CR-1-31B synergized with the oxidative stress inducer, tert-butylhydroquinone (tBHQ), to enhance cell death under oxidative stress. Proteomic analysis revealed a subset of proteins that were upregulated by tBHQ alone, but inhibited by co-treatment of CR-1-31B, most notably the NRF2 antioxidant transcription factor, and NRF2 inactivation phenocopied CR-1-31B in blocking OS lung metastasis in vivo. Collectively, our data reveal that targeting eIF4A1 with CR-1-31B is highly effective in blocking OS metastasis by blunting the NRF2 antioxidant response.
Project description:In this study we looked for the main protein pathway regulators which were responsible for the therapeutic impact on colon cancers when combining magnetic hyperthermia with the chemo-therapeutic agent 5-fluorouracil (5FU). In this context, chitosan-coated magnetic nanoparticles (MNP) functionalized with 5FU were intratumorally injected into subcutaneous human colon cancer xenografts (HT-29) in mice and exposed to an alternating magnetic field. A decreased tu-mor growth was found particularly for the combined thermo-chemotherapy vs. the correspond-ing monotherapies. By using computational analysis of the tumor proteome, we found upregu-lated functional pathway categories termed “cellular stress and injury”, “intracellular second messenger and nuclear receptor signaling”, “immune responses”, and “growth proliferation and development”. We predict TGF-beta, and other mediators, as important upstream regulators. In conclusion our findings show that the combined thermo-chemotherapy induces thrombogenic collagen fibers which are able to impair tumor nutrient supply. Further on, we associate several responses to the recognition of damage associated molecular patterns (DAMPs) by phagocytic cells, which immigrate into the tumor area. The activation of some pathways associated with cell survival implies the necessity to conduct multiple therapy sessions in connection with a corre-sponding monitoring, which could possibly be conducted on the base of the identified protein regulators.
Project description:Purpose: Chemotherapy is pivotal in the multimodal treatment of pancreatic cancer patients. In recent years, technical advances have developed experimental methods that unveiled a high degree of inter- and intratumoral heterogeneity in pancreatic cancer. We hypothesized that intratumoral heterogeneity (ITH) impacts response to gemcitabine treatment and demands specific targeting of resistant subclones. Experimental Design: We addressed the effect of ITH on response to gemcitabine treatment using single cell-derived cell lines (SCDCL) from the classical-like cell line BxPC3 and the basal-like cell line Panc-1 which were analyzed by mRNA-seq and mass spectrometry. Results: Individual SCDCLs of the parental tumor cell populations of BxPC3 and Panc-1 showed considerable heterogeneity in response to gemcitabine. Unsupervised principal component analysis (PCA) including the 1,000 most variably expressed genes showed a clustering of the SCDCLs according to their respective sensitivity to gemcitabine treatment for BxPC3, while this clustering was less clear for Panc-1. In BxPC3 SCDCLs, enriched signaling pathways EMT, TNF signaling via NfKB, and IL2STAT5 signaling correlated with more resistant behavior to gemcitabine. In Panc-1 SCDCLs MYC targets V1 and V2 as well as E2F targets were associated with stronger resistance to gemcitabine. Feature extraction of proteomes again identified less proteins whose expression was associated with the response of individual SCDCLs in Panc-1 compared to BxPC3. Based on molecular profiles, we could show that the gemcitabine-resistant SCDCLs of both BxPC3 and Panc-1 are more sensitive to the BET inhibitor JQ1 compared to the respective gemcitabine-sensitive SCDCLs. Conclusions: Our model system of SCDCLs identified gemcitabine-resistant subclones within a parental tumor population and provides evidence for the critical role of ITH for treatment response in pancreatic cancer. Through molecular profiling, we identified specific signaling pathways and protein signatures that might help to explain the differential response to treatment among clones. We exploited these molecular differences for an improved and more targeted therapy of resistant subclones of a heterogeneous tumor.
Project description:During aging, the regenerative capacity of skeletal muscle decreases due to intrinsic changes in muscle stem cells (MuSCs) and alterations in their niche. Here, we used quantitative mass spectrometry to characterize intrinsic changes in the MuSC proteome and remodeling of the MuSC niche during aging. We generated a network connecting age-affected ligands located in the niche and cell surface receptors on MuSCs. Thereby, we revealed signaling via Integrins, Lrp1, Egfr and Cd44 as the major cell communication axes perturbed through aging. We investigated the effect of Smoc2, a secreted protein that accumulates with aging, originating from fibro-adipogenic progenitors. Increased levels of Smoc2 contribute to the aberrant Itgb1/MAPK signaling observed during aging, thereby causing impaired MuSC functionality and muscle regeneration. By connecting changes in the proteome of MuSCs to alterations of their niche, our work will enable a better understanding of how MuSCs are affected during aging.
Project description:Peptide fragmentation spectra are routinely predicted in the interpretation of mass spectrometry-based proteomics data. Unfortunately, the generation of fragment ions is not well enough understood to estimate fragment ion intensities accurately. Here, we demonstrate that machine learning can predict peptide fragmentation patterns in mass spectrometers with accuracy within the uncertainty of the measurements. Moreover, analysis of our models reveals that peptide fragmentation depends on long-range interactions within a peptide sequence. We illustrate the utility of our models by applying them to the analysis of both data-dependent and data-independent acquisition datasets. In the former case, we observe a significant increase in the total number of peptide identifications at fixed false discovery rate. In the latter case we demonstrate that the use of predicted MS/MS spectra is equivalent to the use of spectra from experimentallibraries, indicating that fragmentation libraries for proteomics are becoming obsolete.
Project description:Hematopoietic stem cell transplantation (HSCT) is successfully applied since the late 1950s, however, its efficacy still needs to be improved. A promising strategy is to transplant high numbers of pluripotent hematopoietic stem cells (HSCs). Therefore, an advanced ex vivo culture system is needed that supports the proliferation and maintains the pluripotency of HSC to override possible limitations in cell numbers gained from donors. To model the natural HSC niche in vitro and thus, to amplify high numbers of undifferentiated HSCs, we used an optimized HSC cell culture medium in combination with artificial 3D bone marrow-like scaffolds made of polydimethylsiloxane (PDMS). After 14 days in vitro (DIV) cell culture, we performed transcriptome and proteome analysis of the whole cell populations. Ingenuity pathway analysis (IPA) indicated that our 3D PDMS cell culture scaffolds activated interleukin, SREBP, mTOR and FOXO signaling pathways as well as the HSC metabolism, which we confirmed by ELISA, Western blot and metabolic flux analysis. These molecular signaling pathways and HSC metabolism are well known to promote the expansion HSCs and are involved in their pluripotency maintenance. After selection and enrichment of immature CD34-positive/CD38-negative HSCs using FACS sorting, we could confirm our findings by another proteome analysis followed by IPA. Thus, we could show that our 3D bone marrow-like PDMS scaffolds activate key molecular signaling pathways to amplify the numbers of undifferentiated HSC efficiently ex vivo.