News | IMPRS for Molecules of Life

Publication of IMPRS-LS student Mahesh Lingaraju

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Lingaraju, M., Johnsen, D., Schlundt, A., Langer, L.M., Basquin, J., Sattler, M., Heick Jensen, T., Falk, S., and Conti, E.
Nat Commun, 2019, 10, 3393.
doi: 10.1038/s41467-019-11339-x

The MTR4 helicase recruits nuclear adaptors of the human RNA exosome using distinct arch-interacting motifs

The nuclear exosome and its essential co-factor, the RNA helicase MTR4, play crucial roles in several RNA degradation pathways. Besides unwinding RNA substrates for exosome-mediated degradation, MTR4 associates with RNA-binding proteins that function as adaptors in different RNA processing and decay pathways. Here, we identify and characterize the interactions of human MTR4 with a ribosome processing adaptor, NVL, and with ZCCHC8, an adaptor involved in the decay of small nuclear RNAs. We show that the unstructured regions of NVL and ZCCHC8 contain short linear motifs that bind the MTR4 arch domain in a mutually exclusive manner. These short sequences diverged from the arch-interacting motif (AIM) of yeast rRNA processing factors. Our results suggest that nuclear exosome adaptors have evolved canonical and non-canonical AIM sequences to target human MTR4 and demonstrate the versatility and specificity with which the MTR4 arch domain can recruit a repertoire of different RNA-binding proteins.

Graduation of Sandra Lemke, Philipp Blumhardt and Mehrshad Pakdel

Congratulations on your PhD!

Sandra Lemke
Analysis of molecular forces transmitted by Talin during muscle development in vivo
RG: Frank Schnorrer

Philipp Blumhardt
Surface-Integrated Fluorescence Correlation Spectroscopy (SI-FCS) for the quantification of transient membrane and surface binding
RG: Petra Schwille

Mehrshad Pakdel
Activity of the SPCA1 calcium ATPase couples sphingomyelin synthesis to sorting of secretory proteins in the trans-Golgi network
RG: Julia von Blume

Publication of IMPRS-LS student Matea Konjevic Sabolek

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Konjevic Sabolek, M., Held, K., Beltran, E., Niedl, A.G., Meinl, E., Hohlfeld, R., Lassmann, H., and Dornmair, K.
Ann Clin Transl Neurol, 2019, 6, 1151-1164
doi: 10.1002/acn3.783

Communication of CD8⁺ T cells with mononuclear phagocytes in multiple sclerosis

OBJECTIVE: CD8⁺ T cells are the most prevailing lymphocyte population in inflammatory lesions of patients with multiple sclerosis (MS) but it is not even known whether they are merely passive bystanders or actively communicate with other cells in the brain. To identify their potential interaction partners, we analyzed CD8⁺ T cells that contained vectorially oriented cytotoxic granules and analyzed the areas to which the granules pointed.
METHODS: We stained cryo-sections of active MS lesions of an index patient with antibodies to CD8 and perforin, searched for vectorially oriented perforin granules, and isolated target areas opposing the granules and control areas by laser-microdissection. From both areas, we analyzed cell-type specific transcripts by next-generation sequencing. In parallel, we stained samples from the index-patient and other patients by four-color immunohistochemistry (IHC).
RESULTS: We found transcripts of the mononuclear phagocyte (MP) specific markers CD163 and CD11b only in the microdissected target areas but not in control areas. We validated the finding that MPs are communication partners of CD8⁺ T cells in MS lesions by classical IHC in samples from the index-patient and other patients with acute and progressive MS and other inflammatory neurological diseases.
INTERPRETATION: Because CD163 and CD11b are specifically expressed in MPs, our findings suggest that CD8⁺ T cells communicate with local MPs. Although it is still unclear if these interactions lead to killing of the communication partners by CD8⁺ T cells, our data underline that CD8⁺ T cells play an active role in the pathogenesis of MS.

Publication of IMPRS-LS student Nena Matscheko

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Matscheko, N., Mayrhofer, P., Rao, Y., Beier, V., and Wollert, T.
PLoS Biol, 2019, 17, e3000377
doi: 10.1371/journal.pbio.3000377

Atg11 tethers Atg9 vesicles to initiate selective autophagy

Autophagy recycles cytoplasmic components by sequestering them in double membrane-surrounded autophagosomes. The two proteins Atg11 and Atg17 are scaffolding components of the Atg1 kinase complex. Atg17 recruits and tethers Atg9-donor vesicles, and the corresponding Atg1 kinase complex induces the formation of nonselective autophagosomes. Atg11 initiates selective autophagy and coordinates the switch to nonselective autophagy by recruiting Atg17. The molecular function of Atg11 remained, however, less well understood. Here, we demonstrate that Atg11 is activated by cargo through a direct interaction with autophagy receptors. Activated Atg11 dimerizes and tethers Atg9 vesicles, which leads to the nucleation of phagophores in direct vicinity of cargo. Starvation reciprocally regulates the activity of both tethering factors by initiating the degradation of Atg11 while Atg17 is activated. This allows Atg17 to sequester and tether Atg9 vesicles independent of cargo to nucleate nonselective phagophores. Our data reveal insights into the molecular mechanisms governing cargo selection and specificity in autophagy.

Graduation of Nicole Teichmann and Sigrun Schmähling

Congratulations on your PhD!

Nicole Teichmann
Preclinical evaluation of an oral MEK1/2 inhibitor as a therapeutic strategy for GEMM-based Pancreatic ductal adenocarcinoma (PDAC)
RG: Jens Siveke

Sigrun Schmähling
Biochemical purification and functional characterization of a novel trithorax-group protein complex
RG: Jürg Müller

Publication of IMPRS-LS student Kimberly Cramer

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Cramer, K., Bolender, A.L., Stockmar, I., Jungmann, R., Kasper, R., and Shin, J.Y.
Int J Mol Sci, 2019, 20, [Epub ahead of print].
doi: 10.3390/ijms20143376

Visualization of Bacterial Protein Complexes Labeled with Fluorescent Proteins and Nanobody Binders for STED Microscopy

In situ visualization of molecular assemblies near their macromolecular scale is a powerful tool to investigate fundamental cellular processes. Super-resolution light microscopies (SRM) overcome the diffraction limit and allow researchers to investigate molecular arrangements at the nanoscale. However, in bacterial cells, visualization of these assemblies can be challenging because of their small size and the presence of the cell wall. Thus, although conceptually promising, successful application of SRM techniques requires careful optimization in labeling biochemistry, fluorescent dye choice, bacterial biology and microscopy to gain biological insights. Here, we apply Stimulated Emission Depletion (STED) microscopy to visualize cell division proteins in bacterial cells, specifically E. coli and B. subtilis. We applied nanobodies that specifically recognize fluorescent proteins, such as GFP, mCherry2 and PAmCherry, fused to targets for STED imaging and evaluated the effect of various organic fluorescent dyes on the performance of STED in bacterial cells. We expect this research to guide scientists for in situ macromolecular visualization using STED in bacterial systems.

Publication of IMPRS-LS student Derya Kabacaoglu

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Kabacaoglu, D., Ruess, D.A., Ai, J., and Algul, H.
Cancers (Basel), 2019, 11.
doi: 10.3390/cancers11070937

NF-kappaB/Rel Transcription Factors in Pancreatic Cancer: Focusing on RelA, c-Rel, and RelB

Regulation of Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)/Rel transcription factors (TFs) is extremely cell-type-specific owing to their ability to act disparately in the context of cellular homeostasis driven by cellular fate and the microenvironment. This is also valid for tumor cells in which every single component shows heterogenic effects. Whereas many studies highlighted a per se oncogenic function for NF-κB/Rel TFs across cancers, recent advances in the field revealed their additional tumor-suppressive nature. Specifically, pancreatic ductal adenocarcinoma (PDAC), as one of the deadliest malignant diseases, shows aberrant canonical-noncanonical NF-κB signaling activity. Although decades of work suggest a prominent oncogenic activity of NF-κB signaling in PDAC, emerging evidence points to the opposite including anti-tumor effects. Considering the dual nature of NF-κB signaling and how it is closely linked to many other cancer related signaling pathways, it is essential to dissect the roles of individual Rel TFs in pancreatic carcinogenesis and tumor persistency and progression. Here, we discuss recent knowledge highlighting the role of Rel TFs RelA, RelB, and c-Rel in PDAC development and maintenance. Next to providing rationales for therapeutically harnessing Rel TF function in PDAC, we compile strategies currently in (pre-)clinical evaluation.

Publication of IMPRS-LS student Maike Kober-Hasslacher

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Kober-Hasslacher, M., and Schmidt-Supprian, M.
Cancers (Basel), 2019, 11.
doi: 10.3390/cancers11070941

The Unsolved Puzzle of c-Rel in B Cell Lymphoma

Aberrant constitutive activation of Rel/NF-κB transcription factors is a hallmark of numerous cancers. Of the five Rel family members, c-Rel has the strongest direct links to tumorigenesis. c-Rel is the only member that can malignantly transform lymphoid cells in vitro. Furthermore, c-Rel is implicated in human B cell lymphoma through the frequent occurrence of REL gene locus gains and amplifications. In normal physiology, high c-Rel expression predominates in the hematopoietic lineage and a diverse range of stimuli can trigger enhanced expression and activation of c-Rel. Both expression and activation of c-Rel are tightly regulated on multiple levels, indicating the necessity to keep its functions under control. In this review we meta-analyze and integrate studies reporting gene locus aberrations to provide an overview on the frequency of REL gains in human B cell lymphoma subtypes, namely follicular lymphoma, diffuse large B cell lymphoma, primary mediastinal B cell lymphoma, and classical Hodgkin lymphoma. We also summarize current knowledge on c-Rel expression and protein localization in these human B cell lymphomas and discuss the co-amplification of BCL11A with REL. In addition, we highlight and illustrate key pathways of c-Rel activation and regulation with a specific focus on B cell biology.

Graduation of Anna-Lena Cost and Philipp Glock

Congratulations on your PhD!

Anna-Lena Cost
Molecular Force Measurements in Desmosomes
RG: Carsten Grashoff

Philipp Glock
Controlling and Reshaping Biological Reaction-Diffusion
RG: Petra Schwille

Publication of IMPRS-LS student Iris Martí Fernandez

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Marti Fernandez, I., Macrini, C., Krumbholz, M., Hensbergen, P.J., Hipgrave Ederveen, A.L., Winklmeier, S., Vural, A., Kurne, A., Jenne, D., Kamp, F., Gerdes, L.A., Hohlfeld, R., Wuhrer, M., Kumpfel, T., and Meinl, E.
Front Immunol, 2019, 10, 1189.
doi: 10.3389/fimmu.2019.01189

The Glycosylation Site of Myelin Oligodendrocyte Glycoprotein Affects Autoantibody Recognition in a Large Proportion of Patients

Autoantibodies to myelin oligodendrocytes glycoprotein (MOG) are found in a fraction of patients with inflammatory demyelination and are detected with MOG-transfected cells. While the prototype anti-MOG mAb 8-18C5 and polyclonal anti-MOG responses from different mouse strains largely recognize the FG loop of MOG, the human anti-MOG response is more heterogeneous and human MOG-Abs recognizing different epitopes were found to be pathogenic. The aim of this study was to get further insight into details of antigen-recognition by human MOG-Abs focusing on the impact of glycosylation. MOG has one known N-glycosylation site at N31 located in the BC loop linking two beta-sheets. We compared the reactivity to wild type MOG with that toward two different mutants in which the neutral asparagine of N31 was mutated to negatively charged aspartate or to the neutral alanine. We found that around 60% of all patients (16/27) showed an altered reactivity to one or both of the mutations. We noted seven different patterns of recognition of the two glycosylation-deficient mutants by different patients. The introduced negative charge at N31 enhanced recognition in some, but reduced recognition in other patients. In 7/27 patients the neutral glycosylation-deficient mutant was recognized stronger. The folding of the extracellular domain of MOG with the formation of beta-sheets did not depend on its glycosylation as seen by circular dichroism. We determined the glycan structure of MOG produced in HEK cells by mass spectrometry. The most abundant glycoforms of MOG expressed in HEK cells are diantennary, contain a core fucose, an antennary fucose, and are decorated with α2,6 linked Neu5Ac, while details of the glycoforms of MOG in myelin remain to be identified. Together, we (1) increase the knowledge about heterogeneity of human autoantibodies to MOG, (2) show that the BC loop affects recognition in about 60% of the patients, (3) report that all patients recognized the unglycosylated protein backbone, while (4) in about 20% of the patients the attached sugar reduces autoantibody binding presumably via steric hindrance. Thus, a neutral glycosylation-deficient mutant of MOG might enhance the sensitivity to identify MOG-Abs.