Bio

Honors & Awards


  • Postdoctoral Research Fellowship, German Research Foundation (DFG)

Professional Education


  • Doctor of Philosophy, Albert Ludwigs Universitat Freiburg (2013)
  • Diploma, Georg August Universitat Gottingen (2009)

Stanford Advisors


Publications

All Publications


  • Impact of Left Ventricular Assist Device Therapy on the Cardiac Proteome and Metabolome Composition in Ischemic Cardiomyopathy. Artificial organs Shahinian, J. H., Rog-Zielinska, E. A., Schlimpert, M., Mayer, B., Tholen, S., Kammerer, B., Biniossek, M. L., Beyersdorf, F., Schilling, O., Siepe, M. 2019

    Abstract

    The changes in the myocardial proteome and metabolome associated with left ventricular assist device (LVAD) therapy in patients with ischemic cardiomyopathy (ICM) are poorly characterized. We investigated the impact of mechanical unloading following LVAD therapy on the myocardial proteome and metabolome.Matched samples of 5 patients' myocardial tissue, harvested at time of LVAD implant ("pre-LVAD") or heart transplant ("post-LVAD") were studied by quantitative proteomics and metabolomics as well as being probed for T-tubule structure and connexin-43 distribution. Moreover, pre-LVAD proteome profiles of ICM context were bioinformatically compared to pre-LVAD proteome profiles of dilate cardiac myopathy (DCM).More than 2120 proteins were reliably identified and quantified in paired patient samples. LVAD therapy led to proteomic remodelling, including reduced levels of ?-1-antichymotrypsin together with an overall decrease of immune response proteins and an increase of proteins involved in membrane biology. Metabolomics highlighted increased glucose and glucose-6-phosphate levels in the left ventricle upon LVAD therapy. Wheat germ agglutinin staining demonstrated improved T-tubule structure. Connexin-43 displayed a trend for more pronounced intercalated disc localization. In comparing pre-LVAD proteome profiles of ICM context with pre-LVAD proteome profiles of dilate cardiac myopathy (DCM) we noticed an overrepresentation in ICM of proteins accosiated with humoral immune response.Our findings underline an impact of LVAD therapy on left ventricular biology in ICM. The proteomic, metabolomic, and structural alterations described here are typically associated with cardiac recovery. On the molecular level, our findings indicate the possibility of cardiac remodeling under LVAD therapy in ICM. This article is protected by copyright. All rights reserved.

    View details for DOI 10.1111/aor.13566

    View details for PubMedID 31494943

  • A Transcriptional Circuit Filters Oscillating Circadian Hormonal Inputs to Regulate Fat Cell Differentiation. Cell metabolism Bahrami-Nejad, Z., Zhao, M. L., Tholen, S., Hunerdosse, D., Tkach, K. E., van Schie, S., Chung, M., Teruel, M. N. 2018; 27 (4): 854?68.e8

    Abstract

    Glucocorticoid and other adipogenic hormones are secreted in mammals in circadian oscillations. Loss of this circadian oscillation pattern correlates with obesity in humans, raising the intriguing question of how hormone secretion dynamics affect adipocyte differentiation. Using live, single-cell imaging of the key adipogenic transcription factors CEBPB and PPARG, endogenously tagged with fluorescent proteins, we show that pulsatile circadian hormone stimuli are rejected by the adipocyte differentiation control system. In striking contrast, equally strong persistent signals trigger maximal differentiation. We identify the mechanism of how hormone oscillations are filtered as a combination of slow and fast positive feedback centered on PPARG. Furthermore, we confirm in mice that flattening of daily glucocorticoid oscillations significantly increases the mass of subcutaneous and visceral fat pads. Together, our study provides a molecular mechanism for why stress, Cushing's disease, and other conditions for which glucocorticoid secretion loses its pulsatility may lead to obesity.

    View details for PubMedID 29617644

  • Identification of Novel Natural Substrates of Fibroblast Activation Protein-alpha by Differential Degradomics and Proteomics. Molecular & cellular proteomics : MCP Zhang, H. E., Hamson, E. J., Tholen, S., Chowdhury, S., Bailey, C. G., Koczorowska, M. M., Lay, A. J., Twigg, S. M., Lee, Q., Roediger, B., Biniossek, M. L., O'Rourke, M., McCaughan, G. W., Keane, F. M., Schilling, O., Gorrell, M. D. 2018

    Abstract

    Fibroblast activation protein-? (FAP) is a cell-surface transmembrane-anchored dimeric protease. This unique, constitutively active serine protease has both dipeptidyl aminopeptidase and endopeptidase activities, and can hydrolyze the post-proline bond. FAP expression is very low in adult organs, but is upregulated by activated fibroblasts in sites of tissue remodeling, including fibrosis, atherosclerosis, arthritis and tumors. To identify the endogenous substrates of FAP, we immortalized primary mouse embryonic fibroblasts (MEFs) from FAP gene knockout embryos and then stably transduced them to express either enzymatically active or inactive FAP. The MEF secretomes were then analyzed using degradomic and proteomic techniques. Terminal amine isotopic labelling of substrates (TAILS)-based degradomics identified cleavage sites in collagens, many other extracellular matrix (ECM) and associated proteins, and lysyl oxidase-like-1, CXCL-5, CSF-1 and C1qT6, that were confirmed in vitro. In addition, differential metabolic labelling coupled with quantitative proteomic analysis also implicated FAP in ECM-cell interactions, as well as with coagulation, metabolism and wound healing associated proteins. Plasma from FAP-deficient mice exhibited slower than wild-type clotting times. This study provides a significant expansion of the substrate repertoire of FAP and provides insight into the physiological and potential pathological roles of this enigmatic protease.

    View details for DOI 10.1074/mcp.RA118.001046

    View details for PubMedID 30257879

  • Proteomics highlights decrease of matricellular proteins in left ventricular assist device therapy?. European journal of cardio-thoracic surgery : official journal of the European Association for Cardio-thoracic Surgery Shahinian, J. H., Mayer, B., Tholen, S., Brehm, K., Biniossek, M. L., Füllgraf, H., Kiefer, S., Heizmann, U., Heilmann, C., Rüter, F., Grapow, M., Reuthebuch, O. T., Eckstein, F., Beyersdorf, F., Schilling, O., Siepe, M. 2017

    Abstract

    We investigated the impact of mechanical unloading with a left ventricular assist device (LVAD) on the myocardial proteome.We collected 11 patient-matched samples of myocardial left ventricular tissue of patients with non-ischaemic dilate cardiomyopathy, harvested at time of LVAD implant ('pre-LVAD') and heart transplant ('post-LVAD'). Samples were studied by quantitative proteomics. Further we performed histological assessment of deposited collagens and immune infiltration in both pre- and post-LVAD samples.A core set of ?>1700 proteins was identified and quantified at a false discovery rate ?<1%. The previously established decrease post-LVAD of alpha-1-antichymotrypsin was corroborated. We noted a post-LVAD decrease of matricellular proteins and proteoglycans such as periostin and versican. Also, proteins of the complement system and precursors of cardiac peptide hormones were decreased post-LVAD. An increase post-LVAD was evident for individual proteins linked to the innate immune response, proteins involved in diverse metabolic pathways, and proteins involved in protein synthesis. Histological analysis did not reveal significant alterations post-LVAD of deposited collagens or immune infiltration. The proteomic data further highlighted a pronounced inter-patient heterogeneity with regards to the impact of LVAD therapy on the left ventricular myocardial proteome. Finally, the proteomic data showed differential proteolytic processing in response to LVAD therapy.Our findings underline a strong impact of LVAD therapy on the left ventricular myocardial proteome. Together with previous studies, protein markers of LVAD therapy such as alpha-1-antichymotrypsin are becoming apparent. Further, matricellular proteins are emerging as important components in response to LVAD therapy.

    View details for DOI 10.1093/ejcts/ezx023

    View details for PubMedID 28329269

  • The papain-like cysteine proteinases NbCysP6 and NbCysP7 are highly processive enzymes with substrate specificities complementary to Nicotiana benthamiana cathepsin B. Biochimica et biophysica acta Paireder, M., Tholen, S., Porodko, A., Biniossek, M. L., Mayer, B., Novinec, M., Schilling, O., Mach, L. 2017; 1865 (4): 444?52

    Abstract

    The tobacco-related plant Nicotiana benthamiana is gaining interest as a versatile host for the production of monoclonal antibodies and other protein therapeutics. However, the susceptibility of plant-derived recombinant proteins to endogenous proteolytic enzymes limits their use as biopharmaceuticals. We have now identified two previously uncharacterized N. benthamiana proteases with high antibody-degrading activity, the papain-like cysteine proteinases NbCysP6 and NbCysP7. Both enzymes are capable of hydrolysing a wide range of synthetic substrates, although only NbCysP6 tolerates basic amino acids in its specificity-determining S2 subsite. The overlapping substrate specificities of NbCysP6 and NbCysP7 are also documented by the closely related properties of their other subsites as deduced from the action of the enzymes on proteome-derived peptide libraries. Notable differences were observed to the substrate preferences of N. benthamiana cathepsin B, another antibody-degrading papain-like cysteine proteinase. The complementary activities of NbCysP6, NbCysP7 and N. benthamiana cathepsin B indicate synergistic roles of these proteases in the turnover of recombinant and endogenous proteins in planta, thus representing a paradigm for the shaping of plant proteomes by the combined action of papain-like cysteine proteinases.

    View details for DOI 10.1016/j.bbapap.2017.02.007

    View details for PubMedID 28188928

  • The death enzyme CP14 is a unique papain-like cysteine proteinase with a pronounced S2 subsite selectivity ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Paireder, M., Mehofer, U., Tholen, S., Porodko, A., Schaehs, P., Maresch, D., Biniossek, M. L., Van der Hoorn, R. A., Lenarcic, B., Novinec, M., Schilling, O., Mach, L. 2016; 603: 110-117

    Abstract

    The cysteine protease CP14 has been identified as a central component of a molecular module regulating programmed cell death in plant embryos. CP14 belongs to a distinct subfamily of papain-like cysteine proteinases of which no representative has been characterized thoroughly to date. However, it has been proposed that CP14 is a cathepsin H-like protease. We have now produced recombinant Nicotiana benthamiana CP14 (NbCP14) lacking the C-terminal granulin domain. As typical for papain-like cysteine proteinases, NbCP14 undergoes rapid autocatalytic activation when incubated at low pH. The mature protease is capable of hydrolysing several synthetic endopeptidase substrates, but cathepsin H-like aminopeptidase activity could not be detected. NbCP14 displays a strong preference for aliphatic over aromatic amino acids in the specificity-determining P2 position. This subsite selectivity was also observed upon digestion of proteome-derived peptide libraries. Notably, the specificity profile of NbCP14 differs from that of aleurain-like protease, the N. benthamiana orthologue of cathepsin H. We conclude that CP14 is a papain-like cysteine proteinase with unusual enzymatic properties which may prove of central importance for the execution of programmed cell death during plant development.

    View details for DOI 10.1016/j.abb.2016.05.017

    View details for Web of Science ID 000379886600013

    View details for PubMedID 27246477

  • Skin Barrier Defects Caused by Keratinocyte-Specific Deletion of ADAM17 or EGFR Are Based on Highly Similar Proteome and Degradome Alterations JOURNAL OF PROTEOME RESEARCH Tholen, S., Wolf, C., Mayer, B., Knopf, J. D., Loeffek, S., Qian, Y., Kizhakkedathu, J. N., Biniossek, M. L., Franzke, C., Schilling, O. 2016; 15 (5): 1402-1417

    Abstract

    Keratinocyte-specific deletion of ADAM17 in mice impairs terminal differentiation of keratinocytes leading to severe epidermal barrier defects. Mice deficient for ADAM17 in keratinocytes phenocopy mice with a keratinocyte-specific deletion of epidermal growth factor receptor (EGFR), which highlights the role of ADAM17 as a "ligand sheddase" of EGFR ligands. In this study, we aim for the first proteomic/degradomic approach to characterize the disruption of the ADAM17-EGFR signaling axis and its consequences for epidermal barrier formation. Proteomic profiling of the epidermal proteome of mice deficient for either ADAM17 or EGFR in keratinocytes at postnatal days 3 and 10 revealed highly similar protein alterations for ADAM17 and EGFR deficiency. These include massive proteome alterations of structural and regulatory components important for barrier formation such as transglutaminases, involucrin, filaggrin, and filaggrin-2. Cleavage site analysis using terminal amine isotopic labeling of substrates revealed increased proteolytic processing of S100 fused-type proteins including filaggrin-2. Alterations in proteolytic processing are supported by altered abundance of numerous proteases upon keratinocyte-specific Adam17 or Egfr deletion, among them kallikreins, cathepsins, and their inhibitors. This study highlights the essential role of proteolytic processing for maintenance of a functional epidermal barrier. Furthermore, it suggests that most defects in formation of the postnatal epidermal barrier upon keratinocyte-specific ADAM17 deletion are mediated via EGFR.

    View details for DOI 10.1021/acs.jproteome.5b00691

    View details for Web of Science ID 000375891200003

    View details for PubMedID 27089454

  • Pregnancy Specific ?-1 Glycoprotein 1 is Expressed in Pancreatic Ductal Adenocarcinoma and its Subcellular Localization Correlates with Overall Survival. Journal of Cancer Shahinian, J. H., Fuellgraf, H., Tholen, S., Mastroianni, J., Knopf, J. D., Kuehs, M., Mayer, B., Schlimpert, M., Kulemann, B., Kuesters, S., Hoeppner, J., Wellner, U. F., Werner, M., Hopt, U. T., Zeiser, R., Bronsert, P., Schilling, O. 2016; 7 (14): 2018?27

    Abstract

    Proteins of the pregnancy specific ?-1 glycoprotein (PSG) family are renowned for their elevated expression during pregnancy. Only few reports have investigated their expression in adenocarcinomas. We studied the expression of PSG1 in pancreatic adenocarcinoma (PDAC). In a cohort of 104 patient samples, immunohistochemical analysis determined PSG1 expression in every specimen. PSG1 was found at apical and cytoplasmic localization or solely at cytoplasmic localization, with the latter case being correlated to shortened median survival (25 vs 11 months, logrank p-value < 0.001). At the same time, enzyme linked immunosorbent assay (ELISA) did not detect elevated PSG1 levels in the plasma of PDAC patients as opposed to the plasma of healthy, non-pregnant control individuals. We also probed the impact of PSG1 expression in a murine tumor model system, using subcutaneous injection of Colo-26 cells into immunocompetent BALB/c mice. Here, tumor growth was not affected by the expression of human PSG1. Our study reaffirms interest into the tumor-contextual biology of PSG proteins.

    View details for DOI 10.7150/jca.15864

    View details for PubMedID 27877217

  • Fibroblast activation protein-alpha, a stromal cell surface protease, shapes key features of cancer associated fibroblasts through proteome and degradome alterations MOLECULAR ONCOLOGY Koczorowska, M. M., Tholen, S., Bucher, F., Lutz, L., Kizhakkedathu, J. N., De Wever, O., Wellner, U. F., Biniossek, M. L., Stahl, A., Lassmann, S., Schilling, O. 2016; 10 (1): 40-58

    Abstract

    Cancer associated fibroblasts (CAFs) constitute an abundant stromal component of most solid tumors. Fibroblast activation protein (FAP) ? is a cell surface protease that is expressed by CAFs. We corroborate this expression profile by immunohistochemical analysis of colorectal cancer specimens. To better understand the tumor-contextual role of FAP?, we investigate how FAP? shapes functional and proteomic features of CAFs using loss- and gain-of function cellular model systems. FAP? activity has a strong impact on the secreted CAF proteome ("secretome"), including reduced levels of anti-angiogenic factors, elevated levels of transforming growth factor (TGF) ?, and an impact on matrix processing enzymes. Functionally, FAP? mildly induces sprout formation by human umbilical vein endothelial cells. Moreover, loss of FAP? leads to a more epithelial cellular phenotype and this effect was rescued by exogenous application of TGF?. In collagen contraction assays, FAP? induced a more contractile cellular phenotype. To characterize the proteolytic profile of FAP?, we investigated its specificity with proteome-derived peptide libraries and corroborated its preference for cleavage carboxy-terminal to proline residues. By "terminal amine labeling of substrates" (TAILS) we explored FAP?-dependent cleavage events. Although FAP? acts predominantly as an amino-dipeptidase, putative FAP? cleavage sites in collagens are present throughout the entire protein length. In contrast, putative FAP? cleavage sites in non-collagenous proteins cluster at the amino-terminus. The degradomic study highlights cell-contextual proteolysis by FAP? with distinct positional profiles. Generally, our findings link FAP? to key aspects of CAF biology and attribute an important role in tumor-stroma interaction to FAP?.

    View details for DOI 10.1016/j.molonc.2015.08.001

    View details for Web of Science ID 000369560300003

    View details for PubMedID 26304112

  • The stromal cell-surface protease fibroblast activation protein-alpha localizes to lipid rafts and is recruited to invadopodia BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH Knopf, J. D., Tholen, S., Koczorowska, M. M., De Weyer, O., Biniossek, M. L., Schilling, O. 2015; 1853 (10): 2515-2525
  • Out-of-frame start codons prevent translation of truncated nucleo-cytosolic cathepsin L in vivo NATURE COMMUNICATIONS Tholen, M., Hillebrand, L. E., Tholen, S., Sedelmeier, O., Arnold, S. J., Reinheckel, T. 2014; 5

    Abstract

    The lysosomal protease cathepsin L has been reported to cleave various functionally important cytosolic or nuclear proteins. To explain nucleo-cytosolic localization of cathepsin L, it has been hypothesized that skipping of the first start codon during translation initiation results in an N-terminally truncated protein lacking the endoplasmic reticulum-import signal. Here we demonstrate that out-of-frame AUGs prevent translation of truncated cathepsin L in cell culture as well as in a new knock-in mouse model. We further evaluate potential roles of nuclear cathepsin L during early embryonic development. Our analysis reveals normal epiblast development of cathepsin L-deficient embryos, but uncovers a pronounced lysosomal storage phenotype in the extra-embryonic tissue of the visceral endoderm. In conclusion, the phenotypes of cathepsin L deficiency can be fully assigned to lack of canonically targeted cathepsin L, while the biogenesis and functionality of nucleo-cytosolic cathepsin L remain elusive.

    View details for DOI 10.1038/ncomms5931

    View details for Web of Science ID 000342984400005

    View details for PubMedID 25222295

  • Understanding fibroblast activation protein (FAP): Substrates, activities, expression and targeting for cancer therapy PROTEOMICS CLINICAL APPLICATIONS Hamson, E. J., Keane, F. M., Tholen, S., Schilling, O., Gorrell, M. D. 2014; 8 (5-6): 454-463

    Abstract

    Fibroblast activation protein (FAP) is best known for its heightened expression in tumour stroma. This atypical serine protease has both dipeptidyl peptidase and endopeptidase activities, cleaving substrates at a post-proline bond. FAP expression is difficult to detect in non-diseased adult organs, but is greatly upregulated in sites of tissue remodelling, which include liver fibrosis, lung fibrosis, atherosclerosis, arthritis, tumours and embryonic tissues. Due to its restricted expression pattern and dual enzymatic activities, FAP is emerging as a unique therapeutic target. However, methods to exploit and target this protease are advancing more rapidly than knowledge of the fundamental biology of FAP. This review highlights this imbalance, emphasising the need to better define the substrate repertoire and expression patterns of FAP to elucidate its role in biological and pathological processes.

    View details for DOI 10.1002/prca.201300095

    View details for Web of Science ID 000337695200016

    View details for PubMedID 24470260

  • Double deficiency of cathepsins B and L results in massive secretome alterations and suggests a degradative cathepsin-MMP axis CELLULAR AND MOLECULAR LIFE SCIENCES Tholen, S., Biniossek, M. L., Gansz, M., Ahrens, T. D., Schlimpert, M., Kizhakkedathu, J. N., Reinheckel, T., Schilling, O. 2014; 71 (5): 899-916

    Abstract

    Endolysosomal cysteine cathepsins functionally cooperate. Cathepsin B (Ctsb) and L (Ctsl) double-knockout mice die 4 weeks after birth accompanied by (autophago-) lysosomal accumulations within neurons. Such accumulations are also observed in mouse embryonic fibroblasts (MEFs) deficient for Ctsb and Ctsl. Previous studies showed a strong impact of Ctsl on the MEF secretome. Here we show that Ctsb alone has only a mild influence on extracellular proteome composition. Protease cleavage sites dependent on Ctsb were identified by terminal amine isotopic labeling of substrates (TAILS), revealing a prominent yet mostly indirect impact on the extracellular proteolytic cleavages. To investigate the cooperation of Ctsb and Ctsl, we performed a quantitative secretome comparison of wild-type MEFs and Ctsb (-/-) Ctsl (-/-) MEFs. Deletion of both cathepsins led to drastic alterations in secretome composition, highlighting cooperative functionality. While many protein levels were decreased, immunodetection corroborated increased levels of matrix metalloproteinase (MMP)-2. Re-expression of Ctsl rescues MMP-2 abundance. Ctsl and to a much lesser extent Ctsb are able to degrade MMP-2 at acidic and neutral pH. Addition of active MMP-2 to the MEF secretome degrades proteins whose levels were also decreased by Ctsb and Ctsl double deficiency. These results suggest a degradative Ctsl-MMP-2 axis, resulting in increased MMP-2 levels upon cathepsin deficiency with subsequent degradation of secreted proteins such as collagen ?-1 (I).

    View details for DOI 10.1007/s00018-013-1406-1

    View details for Web of Science ID 000330963900009

    View details for PubMedID 23811845

  • Proteomic identification of protease cleavage sites: cell-biological and biomedical applications EXPERT REVIEW OF PROTEOMICS Shahinian, H., Tholen, S., Schilling, O. 2013; 10 (5): 421-433

    Abstract

    Proteolysis shapes proteomes by protein degradation or restricted proteolysis, which generates stable cleavage products. Proteolytic (in-)activation of enzymes and cytokines is an essential aspect of the functional proteome status. Proteome-wide identification and quantification of proteolytic processing is accessible by complementary techniques for the focused analysis of protein termini. These innovative strategies are now widely applied and have transformed protease research. Pioneering studies portrayed apoptotic and caspase-dependent cleavage events. Protease-centric investigations focused predominantly on matrix metalloproteinases (MMPs), granzymes and aspartyl and cysteine cathepsins. The first in vivo degradomic studies were performed with mice lacking either cysteine cathepsins or matrix metalloproteinases. Process-centric degradomic analyses investigated infectious processes and mitochondrial import. Peptidomic analyses yielded disease biomarkers representing cleavage fragments from bodily fluids. The diversity of degradomic endeavors illustrates the importance of portraying proteolytic processing in health and disease. The present review provides an overview of the current status of degradomic studies.

    View details for DOI 10.1586/14789450.2013.841547

    View details for Web of Science ID 000330534800009

    View details for PubMedID 24117201

  • Deletion of Cysteine Cathepsins B or L Yields Differential Impacts on Murine Skin Proteome and Degradome MOLECULAR & CELLULAR PROTEOMICS Tholen, S., Biniossek, M. L., Gansz, M., Gomez-Auli, A., Bengsch, F., Noel, A., Kizhakkedathu, J. N., Boerries, M., Busch, H., Reinheckel, T., Schilling, O. 2013; 12 (3): 611-625

    Abstract

    Numerous studies highlight the fact that concerted proteolysis is essential for skin morphology and function. The cysteine protease cathepsin L (Ctsl) has been implicated in epidermal proliferation and desquamation, as well as in hair cycle regulation. In stark contrast, mice deficient in cathepsin B (Ctsb) do not display an overt skin phenotype. To understand the systematic consequences of deleting Ctsb or Ctsl, we determined the protein abundances of >1300 proteins and proteolytic cleavage events in skin samples of wild-type, Ctsb(-/-), and Ctsl(-/-) mice via mass-spectrometry-based proteomics. Both protease deficiencies revealed distinct quantitative changes in proteome composition. Ctsl(-/-) skin revealed increased levels of the cysteine protease inhibitors cystatin B and cystatin M/E, increased cathepsin D, and an accumulation of the extracellular glycoprotein periostin. Immunohistochemistry located periostin predominantly in the hypodermal connective tissue of Ctsl(-/-) skin. The proteomic identification of proteolytic cleavage sites within skin proteins revealed numerous processing sites that are underrepresented in Ctsl(-/-) or Ctsb(-/-) samples. Notably, few of the affected cleavage sites shared the canonical Ctsl or Ctsb specificity, providing further evidence of a complex proteolytic network in the skin. Novel processing sites in proteins such as dermokine and Notch-1 were detected. Simultaneous analysis of acetylated protein N termini showed prototypical mammalian N-alpha acetylation. These results illustrate an influence of both Ctsb and Ctsl on the murine skin proteome and degradome, with the phenotypic consequences of the absence of either protease differing considerably.

    View details for DOI 10.1074/mcp.M112.017962

    View details for Web of Science ID 000317340600006

    View details for PubMedID 23233448

    View details for PubMedCentralID PMC3591655

  • Limited and degradative proteolysis in the context of posttranslational regulatory networks: current technical and conceptional advances Proteases - Structure and Function Tholen, S., Koczorowska, M. M., Lai, Z. W., Dengjel, J., Schilling, O. Springer. 2013
  • A High-Frequency Mutation in Bacillus subtilis: Requirements for the Decryptification of the gudB Glutamate Dehydrogenase Gene JOURNAL OF BACTERIOLOGY Gunka, K., Tholen, S., Gerwig, J., Herzberg, C., Stuelke, J., Commichau, F. M. 2012; 194 (5): 1036-1044

    Abstract

    Common laboratory strains of Bacillus subtilis encode two glutamate dehydrogenases: the enzymatically active protein RocG and the cryptic enzyme GudB that is inactive due to a duplication of three amino acids in its active center. The inactivation of the rocG gene results in poor growth of the bacteria on complex media due to the accumulation of toxic intermediates. Therefore, rocG mutants readily acquire suppressor mutations that decryptify the gudB gene. This decryptification occurs by a precise deletion of one part of the 9-bp direct repeat that causes the amino acid duplication. This mutation occurs at the extremely high frequency of 10(-4). Mutations affecting the integrity of the direct repeat result in a strong reduction of the mutation frequency; however, the actual sequence of the repeat is not essential. The mutation frequency of gudB was not affected by the position of the gene on the chromosome. When the direct repeat was placed in the completely different context of an artificial promoter, the precise deletion of one part of the repeat was also observed, but the mutation frequency was reduced by 3 orders of magnitude. Thus, transcription of the gudB gene seems to be essential for the high frequency of the appearance of the gudB1 mutation. This idea is supported by the finding that the transcription-repair coupling factor Mfd is required for the decryptification of gudB. The Mfd-mediated coupling of transcription to mutagenesis might be a built-in precaution that facilitates the accumulation of mutations preferentially in transcribed genes.

    View details for DOI 10.1128/JB.06470-11

    View details for Web of Science ID 000300530800015

    View details for PubMedID 22178973

  • Contribution of cathepsin L to secretome composition and cleavage pattern of mouse embryonic fibroblasts BIOLOGICAL CHEMISTRY Tholen, S., Biniossek, M. L., Gessler, A., Mueller, S., Weisser, J., Kizhakkedathu, J. N., Reinheckel, T., Schilling, O. 2011; 392 (11): 961-971

    Abstract

    The endolysosomal cysteine endoprotease cathepsin L is secreted from cells in a variety of pathological conditions such as cancer and arthritis. We compared the secretome composition and extracellular proteolytic cleavage events in cell supernatants of cathepsin L-deficient and wild-type mouse embryonic fibroblasts (MEFs). Quantitative proteomic comparison of cell conditioned media indicated that cathepsin L deficiency affects, albeit in a limited manner, the abundances of extracellular matrix (ECM) components, signaling proteins, and further proteases as well as endogenous protease inhibitors. Immunodetection corroborated that cathepsin L deficiency results in decreased abundance of the ECM protein periostin and elevated abundance of matrix metalloprotease (MMP)-2. While mRNA levels of MMP-2 were not affected by cathepsin L ablation, periostin mRNA levels were reduced, potentially indicating a downstream effect. To characterize cathepsin L contribution to extracellular proteolysis, we performed terminal amine isotopic labeling of substrates (TAILS), an N-terminomic technique for the identification and quantification of native and proteolytically generated protein N-termini. TAILS identified >1500 protein N-termini. Cathepsin L deficiency predominantly reduced the magnitude of collagenous cleavage sites C-terminal to a proline residue. This contradicts cathepsin L active site specificity and indicates altered activity of further proteases as a result of cathepsin L ablation.

    View details for DOI 10.1515/BC-2011-162

    View details for Web of Science ID 000295921900004

    View details for PubMedID 21972973

  • SPABBATS: A pathway-discovery method based on Boolean satisfiability that facilitates the characterization of suppressor mutants BMC SYSTEMS BIOLOGY Florez, L. A., Gunka, K., Polania, R., Tholen, S., Stuelke, J. 2011; 5

    Abstract

    Several computational methods exist to suggest rational genetic interventions that improve the productivity of industrial strains. Nonetheless, these methods are less effective to predict possible genetic responses of the strain after the intervention. This problem requires a better understanding of potential alternative metabolic and regulatory pathways able to counteract the targeted intervention.Here we present SPABBATS, an algorithm based on Boolean satisfiability (SAT) that computes alternative metabolic pathways between input and output species in a reconstructed network. The pathways can be constructed iteratively in order of increasing complexity. SPABBATS allows the accumulation of intermediates in the pathways, which permits discovering pathways missed by most traditional pathway analysis methods. In addition, we provide a proof of concept experiment for the validity of the algorithm. We deleted the genes for the glutamate dehydrogenases of the Gram-positive bacterium Bacillus subtilis and isolated suppressor mutant strains able to grow on glutamate as single carbon source. Our SAT approach proposed candidate alternative pathways which were decisive to pinpoint the exact mutation of the suppressor strain.SPABBATS is the first application of SAT techniques to metabolic problems. It is particularly useful for the characterization of metabolic suppressor mutants and can be used in a synthetic biology setting to design new pathways with specific input-output requirements.

    View details for DOI 10.1186/1752-0509-5-5

    View details for Web of Science ID 000286521700001

    View details for PubMedID 21219666

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