Tilman Voss - Academia.edu (original) (raw)

Papers by Tilman Voss

Journal of Pharmacology and Experimental Therapeutics, Dec 20, 2017

The triple-angiokinase inhibitor nintedanib is an orally available, potent, and selective inhibit... more The triple-angiokinase inhibitor nintedanib is an orally available, potent, and selective inhibitor of tumor angiogenesis by blocking the tyrosine kinase activities of vascular endothelial growth factor receptor (VEGFR) 1-3, platelet-derived growth factor receptor (PDGFR)-a and-b, and fibroblast growth factor receptor (FGFR) 1-3. Nintedanib has received regulatory approval as second-line treatment of adenocarcinoma non-small cell lung cancer (NSCLC), in combination with docetaxel. In addition, nintedanib has been approved for the treatment of idiopathic lung fibrosis. Here we report the results from a broad kinase screen that identified additional kinases as targets for nintedanib in the low nanomolar range. Several of these kinases are known to be mutated or overexpressed and are involved in tumor development (discoidin domain receptor family, member 1 and 2, tropomyosin receptor kinase A (TRKA) and C, rearranged during transfection proto-oncogene [RET proto oncogene]), as well as in fibrotic diseases (e.g., DDRs). In tumor cell lines displaying molecular alterations in potential nintedanib targets, the inhibitor demonstrates direct antiproliferative effects: in the NSCLC cell line NCI-H1703 carrying a PDGFRa amplification (ampl.); the gastric cancer cell line KatoIII and the breast cancer cell line MFM223, both driven by a FGFR2 amplification; AN3CA (endometrial carcinoma) bearing a mutated FGFR2; the acute myeloid leukemia cell lines MOLM-13 and MV-4-11-B with FLT3 mutations; and the NSCLC adenocarcinoma LC-2/ad harboring a CCDC6-RET fusion. Potent kinase inhibition does not, however, strictly translate into antiproliferative activity, as demonstrated in the TRKA-dependent cell lines CUTO-3 and KM-12. Importantly, nintedanib treatment of NCI-H1703 tumor xenografts triggered effective tumor shrinkage, indicating a direct effect on the tumor cells in addition to the antiangiogenic effect on the tumor stroma. These findings will be instructive in guiding future genome-based clinical trials of nintedanib.

European journal of biochemistry, Nov 1, 1993

Human interferon omega 1 (IFN-omega 1) was expressed in Spodoptera frugiperda Sf9 insect cells us... more Human interferon omega 1 (IFN-omega 1) was expressed in Spodoptera frugiperda Sf9 insect cells using the baculovirus expression system. Half of the protein purified by immunoaffinity chromatography was shown to be N-glycosylated at the same site as the natural IFN-omega 1. The degree of glycosylation was independent of the expression rate. While natural IFN-omega 1 was shown to carry complex-type oligosaccharides [Adolf, G. R., Maurer-Fogy, I., Kalsner, I. & Cantell, K. (1990) J. Biol. Chem. 265, 9290-9295], the insect cell produced protein which was demonstrated by lectin blot, mass spectroscopy and HPLC analysis to contain only the core oligosaccharide. Two different structures, (Man)2(GlcNAc)2[Fuc] and (Man)3(GlcNAc)2[Fuc] were identified. The fucosylation was identified to be (alpha 1-6)-linked to the core saccharide. Sialic acid residues were clearly absent. IFN-omega 1 expressed in S. frugiperda cells was shown to be partially truncated at the C-terminus by nine residues; its antiviral activity when glycosylated was significantly lower than the activity of IFN-omega 1 produced by Sendai-virus-stimulated leukocytes. Circular dichroism and fluorescence spectroscopy did not reveal any structural differences between glycosylated and nonglycosylated IFN-omega 1. This implies the importance of a complex-type glycosylation for the maximal biological activity of human IFN-omega 1.

FEBS Letters, Mar 9, 1981

The complete amino acid sequence of the major pulmonary surfactant associated glycoprotein SP 28–... more The complete amino acid sequence of the major pulmonary surfactant associated glycoprotein SP 28–36 has been derived from the cDNA and genomic sequences (White et al. 1985; Floros et al. 1986; Sano et al. 1987). The sequences of the human, canine and rat protein are highly homologous. The protein consists of a polypeptide chain with 228 amino acids. The Mr of this protein, when determined by SDS-Page under reducing conditions, is 32–36 kDa. It runs as a fuzzy band because of its glycolsylation. The N-terminal part of the molecule is characterized by a collagen-like sequence. The C-terminal part of the polypeptide chain contains a putative site for the N-linked glycosylation. The first subcomponent C1q of the human complement C1 (Reid 1979) has similar structural features.

European journal of biochemistry, May 1, 1991

The surfactant‐associated protein, protein A, produced by transgenic Chinese hamster ovary cells ... more The surfactant‐associated protein, protein A, produced by transgenic Chinese hamster ovary cells exhibits a heterogeneous population of structures. Electron microscopy reveals lollipop‐shaped monomers consisting of a collagenous triple helix and a globular domain as well as oligomers in which two, three or more protomers are connected by their collagenous stalks. Each protomer consists of three α‐chains (36 kDa) but under non‐reducing conditions few free α‐chains are observed by SDS/PAGE. Instead γ‐components (three chains), γ2 (six chains) and higher components are observed which are derived from intra‐and inter‐protomer disulfide cross‐linking. Complete reduction at low temperature dissociates the oligomers, but preserves the intact structure of monomers as demonstrated by electron microscopy and trypsin digestion. Circular dichroism revealed an unfolding of the collagen triple helices of fully reduced protein A at 26 °C and of the unreduced protein A around 41.5°C. Reoxidation of the fully reduced protein A re‐established mainly the disulfide bonds within the triple helix but not between monomers. Very few higher assembly forms were reformed even at high protein A concentrations. Cellular in vivo systems must possess an efficient assembly mechanism which cannot be simulated by an in vitro system.

Protein Science, Dec 1, 1995

Recently, protease 2A of human rhinovirus 2 (HRV2 2A) was shown to require a zinc ion for the for... more Recently, protease 2A of human rhinovirus 2 (HRV2 2A) was shown to require a zinc ion for the formation of an active enzyme although zinc is not involved mechanistically. The data presented clearly show that the zinc ion bound to a picornaviral-specific motif represents an essential component of the native structure, probably representing a new Zn-binding motif. This structure, containing mostly &strand elements as shown by CD spectroscopy, changes drastically upon removal of zinc. The zinc-depleted form does represent an intermediate with mostly unchanged secondary structure, but not a fully denatured random coil as obtained by guanidinium hydrochloride. This is indicated by the blue-shifted fluorescence spectra and by CD. The native protein exhibited a cooperative phase transition at 53 "C. In contrast, the zinc-depleted form did not show any transition at all, again demonstrating the stabilizing role of the zinc ion. A structural intermediate was observed during thermal and pH denaturation that may represent a molten globule, as suggested by its ANS binding.

Journal of the Pharmaceutical Society of Japan, May 25, 1995

Oncotarget, Mar 3, 2020

Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphomas worldwide ... more Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphomas worldwide and is characterized by a high diversity of genetic and molecular alterations. Chromosomal translocations and mutations leading to deregulated expression of the transcriptional repressor BCL6 occur in a significant fraction of DLBCL patients. An oncogenic role of BCL6 in the initiation of DLBCL has been shown as the constitutive expression of BCL6 in mice recapitulates the pathogenesis of human DLBCL. However, the role of BCL6 in tumor maintenance remains poorly investigated due to the absence of suitable genetic models and limitations of pharmacological inhibitors. Here, we have utilized tetracycline-inducible CRISPR/Cas9 mutagenesis to study the consequences of BCL6 deletion in established DLBCL models in culture and in vivo. We show that BCL6 knockout in SU-DHL-4 cells in vitro results in an anti-proliferative response 4-7 days after Cas9 induction that was characterized by cell cycle (G1) arrest. Conditional BCL6 deletion in established DLBCL tumors in vivo induced a significant tumor growth inhibition with initial tumor stasis followed by slow tumor growth kinetics. Our findings support a role of BCL6 in the maintenance of lymphoma growth and showcase the utility of inducible CRISPR/ Cas9 systems for probing oncogene addiction. Recent comprehensive sequencing studies in a large cohort of DLBCL patients highlight the heterogeneity of alterations including somatic mutations, copy number alterations, and structural variants [2-4]. Among the most frequently rearranged genes are IGH, BCL2, BCL6, and MYC, with 40%, 21%, 19%, and 8% of cases affected, respectively [5-8]. BCL6 is a DNA-binding protein that represses gene transcription in Germinal Center (GC) B-cells through the recruitment of corepressor proteins. In GCs, BCL6 inhibits DNA damage response pathways and thereby prevents cell cycle arrest and apoptosis during class switch recombination and somatic hypermutation required for antibody maturation in B-cells. Subsequent BCL6 downregulation is crucial

American Journal of Respiratory Cell and Molecular Biology, Jun 1, 1992

We have studied the intracellular transport of the pulmonary surfactant SP-C precursor in vitro a... more We have studied the intracellular transport of the pulmonary surfactant SP-C precursor in vitro and in vivo. In order to monitor the route of the SP-C precursor, we constructed various C-terminally truncated forms of SP-C, which were tagged with a sequence derived from the C-terminus of the human c-myc gene (aa 409-419). Expression of these constructs under the control of the SV40 enhancer and the huMT-II promoter in stably transformed Chinese hamster ovary (CHO) cells revealed that the complete precursor molecule is localized mostly in vesicular structures, probably of lysosomal origin. The truncated precursor lacking the last 22 amino acids at the C-terminus (SP-C/Ctag), however, was restricted to the endoplasmic reticulum as shown by immunofluorescence, using antibodies directed against the tag-sequence, the lysosomal enzyme cathepsin D, the enzyme disulfide isomerase, and the Golgi zone. The intracellular localization was substantiated by subcellular fractionation analysis, suggesting that the last 22 amino acids are necessary for intracellular targeting. Furthermore, Triton X-114 extractions from CHO cells revealed a modification of the SP-C precursor. In vitro translation and pulse-chase experiments in the absence or presence of microsomes showed that the modification occurs post-translationally and in a time-dependent manner. Membrane association studies using an SP-C precursor lacking the mature peptide indicated that the modification is of hydrophobic nature but not a thioester-linked fatty acid.

American Journal of Respiratory Cell and Molecular Biology, 1991

The pulmonary surfactant-associated protein SP-A is encoded by presumably two different genes, re... more The pulmonary surfactant-associated protein SP-A is encoded by presumably two different genes, resulting in slightly different amino acid sequences. Both gene products were expressed in Chinese hamster ovary cells. Their macromolecular structure differed significantly. SP-A alpha 3 exhibited a much higher amount of tetrameric to hexameric structures than SP-A alpha 2, for which dimeric structures predominate. These differences may be caused by the higher expression rates of SP-A alpha 3 presumably due to the presence of introns in the sequence. The occurrence of irregular disulfide links between individual oligomeric SP-A molecules composed of alpha 3-chains together with the demonstrated presence of both gene products in natural human SP-A suggest that the subunits of SP-A are heterotrimers of one alpha 2- and two alpha 3-chains.

Biochemical and Biophysical Research Communications, Sep 1, 1985

Mononuclear cells elaborate effector substances which regulate growth and synthetic activities of... more Mononuclear cells elaborate effector substances which regulate growth and synthetic activities of fibroblasts. We have studied the mechanism by which activated mononuclear cell supernatants inhibit collagen production. Activated supernatants were prepared by culturing human peripheral mononuclear cells with phytohemagglutinin. Human fibroblasts were exposed to the activated supernatants and total cellular RNA was isolated. Collagen mRNA levels were measured using a pro alpha [I] probe and poly(A+) mRNA was assayed using polyuridylic acid as the probe. Collagen production was measured as collagenase-digestible radioactivity. The results showed that pro alpha 1 [I] mRNA levels were decreased in cells incubated with the activated mononuclear cell supernatants and that the decrease was dose and time dependent. The reduction in the pro alpha 1 [I] mRNA correlated with the decrease in collagen production. No inhibition in the poly(A+) message was observed. We conclude that inhibition of collagen production by activated mononuclear cell supernatant occurs primarily at the transcriptional level and that the inhibition may be selective to collagen.

European journal of biochemistry, Jul 1, 1991

The C-terminal non-collagenous domain of the surfactant glycoprotein SP-A was shown to be essenti... more The C-terminal non-collagenous domain of the surfactant glycoprotein SP-A was shown to be essential for its correct folding and assembly, as judged by the secretion of various deletion mutants transiently expressed in COS cells. A deletion mutant coding for this domain was successfully secreted while the expression of the collagenous domain only did not lead to any detectable secretion. Deletion mutants lacking small parts of the non-collagenous domain interfered more or less with the correct folding and assembly of the molecule, thus either reducing or inhibiting the secretion. These data suggest that three prefolded non-collagenous domains register and act as a nucleation center for the folding of the collagenous triple helix which proceeds in a zipper-like fashion towards the N-terminus.

European journal of biochemistry, Mar 1, 1985

NC1, the C-terminal non-collagenous globular domain of collagen IV, represents one of the two end... more NC1, the C-terminal non-collagenous globular domain of collagen IV, represents one of the two end regions responsible for the assembly and cross-linking of the extracellular network of basement membrane collagen. Several cDNA clones for the NC1 domain of the alpha 1(IV) collagen chain of mouse have been isolated by using synthetic oligonucleotides as screening probes for mouse libraries. The oligonucleotides were synthesized according to known stretches of the corresponding protein sequence. Sequencing of the overlapping cDNA clones allowed the complete amino acid sequence of the NC1 domain to be deduced as well as the C-terminal 165 amino acid residues of the triple helix. It consists of 229 amino acid residues which comprise two homologous regions with a high content of cysteine. These DNA and protein sequences are compared to the corresponding sequences of other collagens and discussed with respect to their structural and biological significance.

Journal of Molecular Biology, 1984

A computer method for detecting kinks and flexible sites in thread-like molecules was developed. ... more A computer method for detecting kinks and flexible sites in thread-like molecules was developed. The method is based on the determination of the local curvature along the digitized electron micrographs of the molecules. The root-mean-square curvature provided a measure for the local flexibility, which is related to the persistence length. The influence of various error sources was assessed using computer-simulated thread molecules. "Flexibility profiles" showing the variation of flexibility along molecules were calculated for interstitial, basement membrane and intima collagens. Approximately uniform stiffness corresponding to a persistence length of 60 nm was found for the triple helices of interstitial and monomerie intima collagen. A highly flexible site in interstitial collagen could be assigned to a non-triple helical segment in the sequence surrounding the linkage site of the N-terminal propeptide. A flexible site in the triple helix of collagen I corresponds to a segment of the sequence lacking proline residues. Flexibility varies strongly along the collagen IV molecule. This is consistent with the fact tlmt a series of non-triple helical interruptions have already been found in the not yet coml)leted amino acid sequence of basement membrane collagen. Most of the detected flexible sites allow random bending about the mean zero, but in one case, at the border of the 7 S-domain 6f polymeric collagen IV, a flexible site with a i)referential angle of 40 ~ was found.

PDF file - 74K, Binding of E2F1-4 to the promoter of Gpr19. The result of chromatin immunoprecipi... more PDF file - 74K, Binding of E2F1-4 to the promoter of Gpr19. The result of chromatin immunoprecipitation is shown (full size agarose gel).

Lung, Dec 1, 1990

The genes for all three of the bona fide surfactant associated proteins have been cloned, allowin... more The genes for all three of the bona fide surfactant associated proteins have been cloned, allowing their production by recombinant DNA technology. In addition, improved protocols for the isolation of the natural surfactant proteins (NSP) made them available in larger quantities. Whereas, the NSP are often mixtures of allelic variants or functional isomers from gene families, the recombinant proteins (RSP) are obtained as single pure protein species. Antibodies directed against the N/RSP in combination with DNA probes have allowed new approaches to analyze the formation, location, transport, structure and functional capacities of these molecules as well as their interactions with one another and the phospholipids.

Annals of the New York Academy of Sciences, Dec 1, 1985