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Glioblastoma (GBM) is the most common and most aggressive primary brain tumor, with a very high rate of recurrence and a median survival of 15 months after diagnosis. Abundant evidence suggests that a certain sub-population of cancer cells harbors a stem-like phenotype and is likely responsible for disease recurrence, treatment resistance and potentially even for the infiltrative growth of GBM. GBM incidence has been negatively correlated with the serum levels of 25-hydroxy-vitamin D3, while the low pH within tumors has been shown to promote the expression of the vitamin D3-degrading enzyme 24-hydroxylase, encoded by the CYP24A1 gene. Therefore, we hypothesized that calcitriol can specifically target stem-like glioblastoma cells and induce their differentiation. Here, we show, using in vitro limiting dilution assays, quantitative real-time PCR, quantitative proteomics and ex vivo adult organotypic brain slice transplantation cultures, that therapeutic doses of calcitriol, the hormonally active form of vitamin D3, reduce stemness to varying extents in a panel of investigated GSC lines, and that it effectively hinders tumor growth of responding GSCs ex vivo. We further show that calcitriol synergizes with Temozolomide ex vivo to completely eliminate some GSC tumors. These findings indicate that calcitriol carries potential as an adjuvant therapy for a subgroup of GBM patients and should be analyzed in more detail in follow-up studies.
Cell-free therapy using extracellular vesicles (EVs) from adipose-derived mesenchymal stromal/stem cells (ASCs) seems to be a safe and effective therapeutic option to support tissue and organ regeneration. The application of EVs requires particles with a maximum regenerative capability and hypoxic culture conditions as an in vitro preconditioning regimen has been shown to alter the molecular composition of released EVs. Nevertheless, the EV cargo after hypoxic preconditioning has not yet been comprehensively examined. The aim of the present study was the characterization of EVs from hypoxic preconditioned ASCs. We investigated the EV proteome and their effects on renal tubular epithelial cells in vitro. While no effect of hypoxia was observed on the number of released EVs and their protein content, the cargo of the proteins was altered. Proteomic analysis showed 41 increased or decreased proteins, 11 in a statistically significant manner. Furthermore, the uptake of EVs in epithelial cells and a positive effect on oxidative stress in vitro were observed. In conclusion, culture of ASCs under hypoxic conditions was demonstrated to be a promising in vitro preconditioning regimen, which alters the protein cargo and increases the anti-oxidative potential of EVs. These properties may provide new potential therapeutic options for regenerative medicine.
Upon infection with SARS-CoV-2, a variety of changes happen inside the host cell. The virus hijacks host cell pathways for driving its own replication, while the host counteracts with response mechanisms. To gain a comprehensive understanding of COVID-19, caused by SARS-CoV-2 infection, and develop therapeutic strategies, it is crucial to observe these systematic changes in their entirety. In our recent studies, we followed the effects of SARS-CoV-2 infection on the human proteome, which led to the identification of several drugs that abolished viral proliferation in cells.
The integrated stress response (ISR) is a central cellular adaptive program that is activated by diverse stressors including ER stress, hypoxia and nutrient deprivation to orchestrate responses via activating transcription factor 4 (ATF4). We hypothesized that ATF4 is essential for the adaptation of human glioblastoma (GB) cells to the conditions of the tumor microenvironment and is contributing to therapy resistance against chemotherapy. ATF4 induction in GB cells was modulated pharmacologically and genetically and investigated in the context of temozolomide treatment as well as glucose and oxygen deprivation. The relevance of the ISR was analyzed by cell death and metabolic measurements under conditions to approximate aspects of the GB microenvironment. ATF4 protein levels were induced by temozolomide treatment. In line, ATF4 gene suppressed GB cells (ATF4sh) displayed increased cell death and decreased survival after temozolomide treatment. Similar results were observed after treatment with the ISR inhibitor ISRIB. ATF4sh and ISRIB treated GB cells were sensitized to hypoxia-induced cell death. Our experimental study provides evidence for an important role of ATF4 for the adaptation of human GB cells to conditions of the tumor microenvironment characterized by low oxygen and nutrient availability and for the development of temozolomide resistance. Inhibiting the ISR in GB cells could therefore be a promising therapeutic approach.
Background: Alternative polyadenylation (APA) refers to the regulated selection of polyadenylation sites (PASs) in transcripts, which determines the length of their 3′ untranslated regions (3′UTRs). We have recently shown that SRSF3 and SRSF7, two closely related SR proteins, connect APA with mRNA export. The mechanism underlying APA regulation by SRSF3 and SRSF7 remained unknown.
Results: Here we combine iCLIP and 3′-end sequencing and find that SRSF3 and SRSF7 bind upstream of proximal PASs (pPASs), but they exert opposite effects on 3′UTR length. SRSF7 enhances pPAS usage in a concentration-dependent but splicing-independent manner by recruiting the cleavage factor FIP1, generating short 3′UTRs. Protein domains unique to SRSF7, which are absent from SRSF3, contribute to FIP1 recruitment. In contrast, SRSF3 promotes distal PAS (dPAS) usage and hence long 3′UTRs directly by counteracting SRSF7, but also indirectly by maintaining high levels of cleavage factor Im (CFIm) via alternative splicing. Upon SRSF3 depletion, CFIm levels decrease and 3′UTRs are shortened. The indirect SRSF3 targets are particularly sensitive to low CFIm levels, because here CFIm serves a dual function; it enhances dPAS and inhibits pPAS usage by binding immediately downstream and assembling unproductive cleavage complexes, which together promotes long 3′UTRs.
Conclusions; We demonstrate that SRSF3 and SRSF7 are direct modulators of pPAS usage and show how small differences in the domain architecture of SR proteins can confer opposite effects on pPAS regulation.
The discovery of clustered regularly interspaced short palindromic repeats and their associated proteins (Cas) has revolutionized the field of genome and epigenome editing. A number of new methods have been developed to precisely control the function and activity of Cas proteins, including fusion proteins and small-molecule modulators. Proteolysis-targeting chimeras (PROTACs) represent a new concept using the ubiquitin-proteasome system to degrade a protein of interest, highlighting the significance of chemically induced protein-E3 ligase interaction in drug discovery. Here, we engineered Cas proteins (Cas9, dCas9, Cas12, and Cas13) by inserting a Phe-Cys-Pro-Phe (FCPF) amino acid sequence (known as the π-clamp system) and demonstrate that the modified CasFCPF proteins can be (1) labeled in live cells by perfluoroaromatics carrying the fluorescein or (2) degraded by a perfluoroaromatics-functionalized PROTAC (PROTAC-FCPF). A proteome-wide analysis of PROTAC-FCPF-mediated Cas9FCPF protein degradation revealed a high target specificity, suggesting a wide range of applications of perfluoroaromatics-induced proximity in the regulation of stability, activity, and functionality of any FCPF-tagging protein.
The measurement of protein dynamics by proteomics to study cell remodeling has seen increased attention over the last years. This development is largely driven by a number of technological advances in proteomics methods. Pulsed stable isotope labeling in cell culture (SILAC) combined with tandem mass tag (TMT) labeling has evolved as a gold standard for profiling protein synthesis and degradation. While the experimental setup is similar to typical proteomics experiments, the data analysis proves more difficult: After peptide identification through search engines, data extraction requires either custom scripted pipelines or tedious manual table manipulations to extract the TMT-labeled heavy and light peaks of interest. To overcome this limitation, which deters researchers from using protein dynamic proteomics, we developed a user-friendly, browser-based application that allows easy and reproducible data analysis without the need for scripting experience. In addition, we provide a python package that can be implemented in established data analysis pipelines. We anticipate that this tool will ease data analysis and spark further research aimed at monitoring protein translation and degradation by proteomics.
Oncogenic transformation of lung epithelial cells is a multi-step process, frequently starting with the inactivation of tumor suppressors and subsequent activating mutations in proto-oncogenes, such as members of the PI3K or MAPK family. Cells undergoing transformation have to adjust to changes, such as metabolic requirements. This is achieved, in part, by modulating the protein abundance of transcription factors, which manifest these adjustments. Here, we report that the deubiquitylase USP28 enables oncogenic reprogramming by regulating the protein abundance of proto-oncogenes, such as c-JUN, c-MYC, NOTCH and ΔNP63, at early stages of malignant transformation. USP28 is increased in cancer compared to normal cells due to a feed-forward loop, driven by increased amounts of oncogenic transcription factors, such as c-MYC and c-JUN. Irrespective of oncogenic driver, interference with USP28 abundance or activity suppresses growth and survival of transformed lung cells. Furthermore, inhibition of USP28 via a small molecule inhibitor reset the proteome of transformed cells towards a ‘pre-malignant’ state, and its inhibition cooperated with clinically established compounds used to target EGFRL858R, BRAFV600E or PI3KH1047R driven tumor cells. Targeting USP28 protein abundance already at an early stage via inhibition of its activity therefore is a feasible strategy for the treatment of early stage lung tumours and the observed synergism with current standard of care inhibitors holds the potential for improved targeting of established tumors.