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- ItemThe mTOR and PP2A pathways regulate PHD2 phosphorylation to Fine-Tune HIF1α levels and colorectal cancer cell survival under hypoxia(Amsterdam : Elsevier, 2017) Di Conza, Giusy; Cafarello, Sarah Trusso; Loroch, Stefan; Mennerich, Daniela; Deschoemaeker, Sofie; Di Matteo, Mario; Ehling, Manuel; Gevaert, Kris; Prenen, Hans; Zahedi, Rene Peiman; Sickmann, Albert; Kietzmann, Thomas; Moretti, Fabiola; Mazzone, MassimilianoOxygen-dependent HIF1α hydroxylation and degradation are strictly controlled by PHD2. In hypoxia, HIF1α partly escapes degradation because of low oxygen availability. Here, we show that PHD2 is phosphorylated on serine 125 (S125) by the mechanistic target of rapamycin (mTOR) downstream kinase P70S6K and that this phosphorylation increases its ability to degrade HIF1α. mTOR blockade in hypoxia by REDD1 restrains P70S6K and unleashes PP2A phosphatase activity. Through its regulatory subunit B55α, PP2A directly dephosphorylates PHD2 on S125, resulting in a further reduction of PHD2 activity that ultimately boosts HIF1α accumulation. These events promote autophagy-mediated cell survival in colorectal cancer (CRC) cells. B55α knockdown blocks neoplastic growth of CRC cells in vitro and in vivo in a PHD2-dependent manner. In patients, CRC tissue expresses higher levels of REDD1, B55α, and HIF1α but has lower phospho-S125 PHD2 compared with a healthy colon. Our data disclose a mechanism of PHD2 regulation that involves the mTOR and PP2A pathways and controls tumor growth.
- ItemIdentification of herbal teas and their compounds eliciting antiviral activity against SARS-CoV-2 in vitro(Heidelberg : Springer, 2022) Le-Trilling, Vu Thuy Khanh; Mennerich, Denise; Schuler, Corinna; Sakson, Roman; Lill, Julia K.; Kasarla, Siva Swapna; Kopczynski, Dominik; Loroch, Stefan; Flores-Martinez, Yulia; Katschinski, Benjamin; Wohlgemuth, Kerstin; Gunzer, Matthias; Meyer, Folker; Phapale, Prasad; Dittmer, Ulf; Sickmann, Albert; Trilling, MirkoBackground: The SARS-CoV-2/COVID-19 pandemic has inflicted medical and socioeconomic havoc, and despite the current availability of vaccines and broad implementation of vaccination programs, more easily accessible and cost-effective acute treatment options preventing morbidity and mortality are urgently needed. Herbal teas have historically and recurrently been applied as self-medication for prophylaxis, therapy, and symptom alleviation in diverse diseases, including those caused by respiratory viruses, and have provided sources of natural products as basis for the development of therapeutic agents. To identify affordable, ubiquitously available, and effective treatments, we tested herbs consumed worldwide as herbal teas regarding their antiviral activity against SARS-CoV-2. Results: Aqueous infusions prepared by boiling leaves of the Lamiaceae perilla and sage elicit potent and sustained antiviral activity against SARS-CoV-2 when applied after infection as well as prior to infection of cells. The herbal infusions exerted in vitro antiviral effects comparable to interferon-β and remdesivir but outperformed convalescent sera and interferon-α2 upon short-term treatment early after infection. Based on protein fractionation analyses, we identified caffeic acid, perilla aldehyde, and perillyl alcohol as antiviral compounds. Global mass spectrometry (MS) analyses performed comparatively in two different cell culture infection models revealed changes of the proteome upon treatment with herbal infusions and provided insights into the mode of action. As inferred by the MS data, induction of heme oxygenase 1 (HMOX-1) was confirmed as effector mechanism by the antiviral activity of the HMOX-1-inducing compounds sulforaphane and fraxetin. Conclusions: In conclusion, herbal teas based on perilla and sage exhibit antiviral activity against SARS-CoV-2 including variants of concern such as Alpha, Beta, Delta, and Omicron, and we identified HMOX-1 as potential therapeutic target. Given that perilla and sage have been suggested as treatment options for various diseases, our dataset may constitute a valuable resource also for future research beyond virology.
- ItemMalignant transformation in a defined genetic background: Proteome changes displayed by 2D-PAGE(London : BioMed Central, 2010) Pütz, Stephanie M.; Vogiatzi, Fotini; Stiewe, Thorsten; Sickmann, AlbertBackground: Cancer arises from normal cells through the stepwise accumulation of genetic alterations. Cancer development can be studied by direct genetic manipulation within experimental models of tumorigenesis. Thereby, confusion by the genetic heterogeneity of patients can be circumvented. Moreover, identification of the critical changes that convert a pre-malignant cell into a metastatic, therapy resistant tumor cell, however, is one necessary step to develop effective and selective anti-cancer drugs. Thus, for the current study a cell culture model for malignant transformation was used: Primary human fibroblasts of the BJ strain were sequentially transduced with retroviral vectors encoding the genes for hTERT (cell line BJ-T), simian virus 40 early region (SV40 ER, cell line BJ-TE) and H-Ras V12 (cell line BJ-TER). Results: The stepwise malignant transformation of human fibroblasts was analyzed on the protein level by differential proteome analysis. We observed 39 regulated protein spots and therein identified 67 different proteins. The strongest change of spot patterns was detected due to integration of SV40 ER. Among the proteins being significantly regulated during the malignant transformation process well known proliferating cell nuclear antigen (PCNA) as well as the chaperones mitochondrial heat shock protein 75 kDa (TRAP-1) and heat shock protein HSP90 were identified. Moreover, we find out, that TRAP-1 is already up-regulated by means of SV40 ER expression instead of H-Ras V12. Furthermore Peroxiredoxin-6 (PRDX6), Annexin A2 (p36), Plasminogen activator inhibitor 2 (PAI-2) and Keratin type II cytoskeletal 7 (CK-7) were identified to be regulated. For some protein candidates we confirmed our 2D-PAGE results by Western Blot. Conclusion: These findings give further hints for intriguing interactions between the p16-RB pathway, the mitochondrial chaperone network and the cytoskeleton. In summary, using a cell culture model for malignant transformation analyzed with 2D-PAGE, proteome and cellular changes can be related to defined steps of tumorigenesis
- ItemInteraction of proteins identified in human thyroid cells(Basel : MDPI, 2013) Pietsch, Jessica; Riwaldt, Stefan; Bauer, Johann; Sickmann, Albert; Weber, Gerhard; Grosse, Jirka; Infanger, Manfred; Eilles, Christoph; Grimm, DanielaInfluence of gravity forces on the regulation of protein expression by healthy and malignant thyroid cells was studied with the aim to identify protein interactions. Western blot analyses of a limited number of proteins suggested a time-dependent regulation of protein expression by simulated microgravity. After applying free flow isoelectric focusing and mass spectrometry to search for differently expressed proteins by thyroid cells exposed to simulated microgravity for three days, a considerable number of candidates for gravi-sensitive proteins were detected. In order to show how proteins sensitive to microgravity could directly influence other proteins, we investigated all polypeptide chains identified with Mascot scores above 100, looking for groups of interacting proteins. Hence, UniProtKB entry numbers of all detected proteins were entered into the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) and processed. The program indicated that we had detected various groups of interacting proteins in each of the three cell lines studied. The major groups of interacting proteins play a role in pathways of carbohydrate and protein metabolism, regulation of cell growth and cell membrane structuring. Analyzing these groups, networks of interaction could be established which show how a punctual influence of simulated microgravity may propagate via various members of interaction chains.
- ItemThe bruchpilot cytomatrix determines the size of the readily releasable pool of synaptic vesicles(London : Nature Publishing Group, 2013) Matkovic, Tanja; Siebert, Matthias; Knoche, Elena; Depner, Harald; Mertel, Sara; Owald, David; Schmidt, Manuela; Thomas, Ulrich; Sickmann, Albert; Kamin, Dirk; Hell, Stefan W.; Bürger, Jörg; Hollmann, Christina; Mielke, Thorsten; Wichmann, Carolin; Sigrist, Stephan J.Synaptic vesicles (SVs) fuse at a specialized membrane domain called the active zone (AZ), covered by a conserved cytomatrix. How exactly cytomatrix components intersect with SV release remains insufficiently understood. We showed previously that loss of the Drosophila melanogaster ELKS family protein Bruchpilot (BRP) eliminates the cytomatrix (T bar) and declusters Ca2+ channels. In this paper, we explored additional functions of the cytomatrix, starting with the biochemical identification of two BRP isoforms. Both isoforms alternated in a circular array and were important for proper T-bar formation. Basal transmission was decreased in isoform-specific mutants, which we attributed to a reduction in the size of the readily releasable pool (RRP) of SVs. We also found a corresponding reduction in the number of SVs docked close to the remaining cytomatrix. We propose that the macromolecular architecture created by the alternating pattern of the BRP isoforms determines the number of Ca2+ channel-coupled SV release slots available per AZ and thereby sets the size of the RRP.
- ItemCurrent strategies and findings in clinically relevant post-translational modification-specific proteomics(Milton Park : Taylor & Francis, 2015) Pagel, Oliver; Loroch, Stefan; Sickmann, Albert; Zahedi, René P.Mass spectrometry-based proteomics has considerably extended our knowledge about the occurrence and dynamics of protein post-translational modifications (PTMs). So far, quantitative proteomics has been mainly used to study PTM regulation in cell culture models, providing new insights into the role of aberrant PTM patterns in human disease. However, continuous technological and methodical developments have paved the way for an increasing number of PTM-specific proteomic studies using clinical samples, often limited in sample amount. Thus, quantitative proteomics holds a great potential to discover, validate and accurately quantify biomarkers in body fluids and primary tissues. A major effort will be to improve the complete integration of robust but sensitive proteomics technology to clinical environments. Here, we discuss PTMs that are relevant for clinical research, with a focus on phosphorylation, glycosylation and proteolytic cleavage; furthermore, we give an overview on the current developments and novel findings in mass spectrometry-based PTM research.
- ItemCompomics-utilities: An open-source Java library for computational proteomics(London : BioMed Central, 2011) Barsnes, Harald; Vaudel, Marc; Helsens, Kenny; Sickmann, Albert; Walther, Dirk; Berven, Frode S.The growing interest in the field of proteomics has increased the demand for software tools and applications that process and analyze the resulting data. And even though the purpose of these tools can vary significantly, they usually share a basic set of features, including the handling of protein and peptide sequences, the visualization of (and interaction with) spectra and chromatograms, and the parsing of results from various proteomics search engines. Developers typically spend considerable time and effort implementing these support structures, which detracts from working on the novel aspects of their tool.
- ItemA Homozygous PPP1R21 Splice Variant Associated with Severe Developmental Delay, Absence of Speech, and Muscle Weakness Leads to Activated Proteasome Function(Totowa, NJ : Humana Press, 2023) Hentschel, Andreas; Meyer, Nancy; Kohlschmidt, Nicolai; Groß, Claudia; Sickmann, Albert; Schara-Schmidt, Ulrike; Förster, Fabian; Töpf, Ana; Christiansen, Jon; Horvath, Rita; Vorgerd, Matthias; Thompson, Rachel; Polaparapu, Kiran; Lochmüller, Hanns; Preusse, Corinna; Hannappel, Luis; Schänzer, Anne; Grüneboom, Anika; Gangfuß, Andrea; Roos, AndreasPPP1R21 acts as a co-factor for protein phosphatase 1 (PP1), an important serine/threonine phosphatase known to be essential for cell division, control of glycogen metabolism, protein synthesis, and muscle contractility. Bi-allelic pathogenic variants in PPP1R21 were linked to a neurodevelopmental disorder with hypotonia, facial dysmorphism, and brain abnormalities (NEDHFBA) with pediatric onset. Functional studies unraveled impaired vesicular transport as being part of PPP1R21-related pathomechanism. To decipher further the pathophysiological processes leading to the clinical manifestation of NEDHFBA, we investigated the proteomic signature of fibroblasts derived from the first NEDHFBA patient harboring a splice-site mutation in PPP1R21 and presenting with a milder phenotype. Proteomic findings and further functional studies demonstrate a profound activation of the ubiquitin–proteasome system with presence of protein aggregates and impact on cellular fitness and moreover suggest a cross-link between activation of the proteolytic system and cytoskeletal architecture (including filopodia) as exemplified on paradigmatic proteins including actin, thus extending the pathophysiological spectrum of the disease. In addition, the proteomic signature of PPP1R21-mutant fibroblasts displayed a dysregulation of a variety of proteins of neurological relevance. This includes increase proteins which might act toward antagonization of cellular stress burden in terms of pro-survival, a molecular finding which might accord with the presentation of a milder phenotype of our NEDHFBA patient.
- ItemLUBAC assembles a ubiquitin signaling platform at mitochondria for signal amplification and transport of NF-κB to the nucleus(Hoboken, NJ [u.a.] : Wiley, 2022) Wu, Zhixiao; Berlemann, Lena A.; Bader, Verian; Sehr, Dominik A.; Dawin, Eva; Covallero, Alberto; Meschede, Jens; Angersbach, Lena; Showkat, Cathrin; Michaelis, Jonas B.; Münch, Christian; Rieger, Bettina; Namgaladze, Dmitry; Herrera, Maria Georgina; Fiesel, Fabienne C.; Springer, Wolfdieter; Mendes, Marta; Stepien, Jennifer; Barkovits, Katalin; Marcus, Katrin; Sickmann, Albert; Dittmar, Gunnar; Busch, Karin B.; Riedel, Dietmar; Brini, Marisa; Tatzelt, Jörg; Cali, Tito; Winklhofer, Konstanze F.Mitochondria are increasingly recognized as cellular hubs to orchestrate signaling pathways that regulate metabolism, redox homeostasis, and cell fate decisions. Recent research revealed a role of mitochondria also in innate immune signaling; however, the mechanisms of how mitochondria affect signal transduction are poorly understood. Here, we show that the NF-κB pathway activated by TNF employs mitochondria as a platform for signal amplification and shuttling of activated NF-κB to the nucleus. TNF treatment induces the recruitment of HOIP, the catalytic component of the linear ubiquitin chain assembly complex (LUBAC), and its substrate NEMO to the outer mitochondrial membrane, where M1- and K63-linked ubiquitin chains are generated. NF-κB is locally activated and transported to the nucleus by mitochondria, leading to an increase in mitochondria-nucleus contact sites in a HOIP-dependent manner. Notably, TNF-induced stabilization of the mitochondrial kinase PINK1 furthermore contributes to signal amplification by antagonizing the M1-ubiquitin-specific deubiquitinase OTULIN. Overall, our study reveals a role for mitochondria in amplifying TNF-mediated NF-κB activation, both serving as a signaling platform, as well as a transport mode for activated NF-κB to the nuclear.