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Publication of IMPRS-LS student Lisa Vincenz-Donnelly

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Vincenz-Donnelly, L., Holthusen, H., Korner, R., Hansen, E.C., Presto, J., Johansson, J., Sawarkar, R., Hartl, F.U., and Hipp, M.S.
EMBO J, 2017, [Epub ahead of print].
doi 10.15252/embj.201695841

High capacity of the endoplasmic reticulum to prevent secretion and aggregation of amyloidogenic proteins

Protein aggregation is associated with neurodegeneration and various other pathologies. How specific cellular environments modulate the aggregation of disease proteins is not well understood. Here, we investigated how the endoplasmic reticulum (ER) quality control system handles β-sheet proteins that were designed de novo to form amyloid-like fibrils. While these proteins undergo toxic aggregation in the cytosol, we find that targeting them to the ER (ER-β) strongly reduces their toxicity. ER-β is retained within the ER in a soluble, polymeric state, despite reaching very high concentrations exceeding those of ER-resident molecular chaperones. ER-β is not removed by ER-associated degradation (ERAD) but interferes with ERAD of other proteins. These findings demonstrate a remarkable capacity of the ER to prevent the formation of insoluble β-aggregates and the secretion of potentially toxic protein species. Our results also suggest a generic mechanism by which proteins with exposed β-sheet structure in the ER interfere with proteostasis.

 

Publication of IMPRS-LS student Sahradha Albert

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Albert, S., Schaffer, M., Beck, F., Mosalaganti, S., Asano, S., Thomas, H.F., Plitzko, J.M., Beck, M., Baumeister, W., and Engel, B.D.
Proc Natl Acad Sci USA, 2017, [Epub ahead of print].
doi: 10.1073/pnas.1716305114

Proteasomes tether to two distinct sites at the nuclear pore complex.

The partitioning of cellular components between the nucleus and cytoplasm is the defining feature of eukaryotic life. The nuclear pore complex (NPC) selectively gates the transport of macromolecules between these compartments, but it is unknown whether surveillance mechanisms exist to reinforce this function. By leveraging in situ cryo-electron tomography to image the native cellular environment of Chlamydomonas reinhardtii, we observed that nuclear 26S proteasomes crowd around NPCs. Through a combination of subtomogram averaging and nanometer-precision localization, we identified two classes of proteasomes tethered via their Rpn9 subunits to two specific NPC locations: binding sites on the NPC basket that reflect its eightfold symmetry and more abundant binding sites at the inner nuclear membrane that encircle the NPC. These basket-tethered and membrane-tethered proteasomes, which have similar substrate-processing state frequencies as proteasomes elsewhere in the cell, are ideally positioned to regulate transcription and perform quality control of both soluble and membrane proteins transiting the NPC.

 

Publication of IMPRS-LS student Juliane Jäpel

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Jaepel, J., Hubener, M., Bonhoeffer, T., and Rose, T.
Nat Neurosci, 2017, 20, 1708-1714.
doi: 10.1038/s41593-017-0021-0

Lateral geniculate neurons projecting to primary visual cortex show ocular dominance plasticity in adult mice.

Experience-dependent plasticity in the mature visual system is widely considered to be cortical. Using chronic two-photon Ca2+ imaging of thalamic afferents in layer 1 of binocular visual cortex, we provide evidence against this tenet: the respective dorsal lateral geniculate nucleus (dLGN) cells showed pronounced ocular dominance (OD) shifts after monocular deprivation in adult mice. Most (86%), but not all, of dLGN cell boutons were monocular during normal visual experience. Following deprivation, initially deprived-eye-dominated boutons reduced or lost their visual responsiveness to that eye and frequently became responsive to the non-deprived eye. This cannot be explained by eye-specific cortical changes propagating to dLGN via cortico-thalamic feedback because the shift in dLGN responses was largely resistant to cortical inactivation using the GABAA receptor agonist muscimol. Our data suggest that OD shifts observed in the binocular visual cortex of adult mice may at least partially reflect plasticity of eye-specific inputs onto dLGN neurons.

 

Publication of IMPRS-LS student Annika Frauenstein

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Frauenstein, A., and Meissner, F.
Methods Mol Biol, 2018, 1714, 215-227.
doi: 10.1007/978-1-4939-7519-8_14

Quantitative Proteomics of Secreted Proteins.

Secreted proteins such as cytokines, interleukins, growth factors, and hormones have pleiotropic functions and facilitate intercellular communication in organisms. Quantification of these proteins conventionally relies on antibody-based methods, i.e., enzyme-linked immunosorbent assays (ELISA), whose large-scale use is limited by availability, specificity, and affordability.Here, we describe an experimental and bioinformatics workflow to comprehensively quantify cellular protein secretion by mass spectrometry. Secreted proteins are collected in vitro or ex vivo, digested with proteases and the resulting peptide mixtures are analyzed in single liquid chromatography-mass spectrometry (LC-MS/MS) runs. Label-free quantification and bioinformatics analysis is conducted in the MaxQuant and Perseus computational environment. Our workflow allows the quantification of thousands of secreted proteins spanning a concentration range of four orders of magnitude and permits the systems-level characterization of secretory programs as well as the discovery of proteins with unexpected extracellular functions.

 

Publication of IMPRS-LS student Sara Gutiérrez-Ángel

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Hosp, F.*, Gutierrez-Angel, S.*, Schaefer, M.H., Cox, J., Meissner, F., Hipp, M.S., Hartl, F.U., Klein, R., Dudanova, I., and Mann, M.
Cell Rep, 2017, 21, 2291-2303.
* equal contribution

Spatiotemporal Proteomic Profiling of Huntington's Disease Inclusions Reveals Widespread Loss of Protein Function.

Aggregation of polyglutamine-expanded huntingtin exon 1 (HttEx1) in Huntington's disease (HD) proceeds from soluble oligomers to late-stage inclusions. The nature of the aggregates and how they lead to neuronal dysfunction is not well understood. We employed mass spectrometry (MS)-based quantitative proteomics to dissect spatiotemporal mechanisms of neurodegeneration using the R6/2 mouse model of HD. Extensive remodeling of the soluble brain proteome correlated with insoluble aggregate formation during disease progression. In-depth and quantitative characterization of the aggregates uncovered an unprecedented complexity of several hundred proteins. Sequestration to aggregates depended on protein expression levels and sequence features such as low-complexity regions or coiled-coil domains. In a cell-based HD model, overexpression of a subset of the sequestered proteins in most cases rescued viability and reduced aggregate size. Our spatiotemporally resolved proteome resource of HD progression indicates that widespread loss of cellular protein function contributes to aggregate-mediated toxicity.

 

Publication of IMPRS-LS student Katja Weckmann

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Weckmann, K., Deery, M.J., Howard, J.A., Feret, R., Asara, J.M., Dethloff, F., Filiou, M.D., Iannace, J., Labermaier, C., Maccarrone, G., Webhofer, C., Teplytska, L., Lilley, K., Muller, M.B., and Turck, C.W.
Sci Rep, 2017, 7, 15788.

Ketamine's antidepressant effect is mediated by energy metabolism and antioxidant defense system.

Fewer than 50% of all patients with major depressive disorder (MDD) treated with currently available antidepressants (ADs) show full remission. Moreover, about one third of the patients suffering from MDD does not respond to conventional ADs and develop treatment-resistant depression (TRD). Ketamine, a non-competitive, voltage-dependent N-Methyl-D-aspartate receptor (NMDAR) antagonist, has been shown to have a rapid antidepressant effect, especially in patients suffering from TRD. Hippocampi of ketamine-treated mice were analysed by metabolome and proteome profiling to delineate ketamine treatment-affected molecular pathways and biosignatures. Our data implicate mitochondrial energy metabolism and the antioxidant defense system as downstream effectors of the ketamine response. Specifically, ketamine tended to downregulate the adenosine triphosphate (ATP)/adenosine diphosphate (ADP) metabolite ratio which strongly correlated with forced swim test (FST) floating time. Furthermore, we found increased levels of enzymes that are part of the ‘oxidative phosphorylation’ (OXPHOS) pathway. Our study also suggests that ketamine causes less protein damage by rapidly decreasing reactive oxygen species (ROS) production and lend further support to the hypothesis that mitochondria have a critical role for mediating antidepressant action including the rapid ketamine response.

 

Publication of IMPRS-LS student Georgia Balsevich

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Balsevich, G., Hausl, A.S., Meyer, C.W., Karamihalev, S., Feng, X., Pohlmann, M.L., Dournes, C., Uribe-Marino, A., Santarelli, S., Labermaier, C., Hafner, K., Mao, T., Breitsamer, M., Theodoropoulou, M., Namendorf, C., Uhr, M., Paez-Pereda, M., Winter, G., Hausch, F., Chen, A., Tschop, M.H., Rein, T., Gassen, N.C., and Schmidt, M.V.
Nat Commun, 2017, 8, 1725.

Stress-responsive FKBP51 regulates AKT2-AS160 signaling and metabolic function.

The co-chaperone FKBP5 is a stress-responsive protein-regulating stress reactivity, and its genetic variants are associated with T2D related traits and other stress-related disorders. Here we show that FKBP51 plays a role in energy and glucose homeostasis. Fkbp5 knockout (51KO) mice are protected from high-fat diet-induced weight gain, show improved glucose tolerance and increased insulin signaling in skeletal muscle. Chronic treatment with a novel FKBP51 antagonist, SAFit2, recapitulates the effects of FKBP51 deletion on both body weight regulation and glucose tolerance. Using shorter SAFit2 treatment, we show that glucose tolerance improvement precedes the reduction in body weight. Mechanistically, we identify a novel association between FKBP51 and AS160, a substrate of AKT2 that is involved in glucose uptake. FKBP51 antagonism increases the phosphorylation of AS160, increases glucose transporter 4 expression at the plasma membrane, and ultimately enhances glucose uptake in skeletal myotubes. We propose FKBP51 as a mediator between stress and T2D development, and potential target for therapeutic approaches.

 

Publication of IMPRS-LS student Vladimir Thoma

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Thoma V., Kobayashi K. and Tanimoto H.
eNeuro 4, 2017

The Role of the Gustatory System in the Coordination of Feeding

To survive, all animals must find, inspect and ingest food. Behavioral coordination and control of feeding is therefore a challenge that animals must face. Here, we focus on how the gustatory system guides the precise execution of behavioral sequences that promote ingestion and suppresses competing behaviors. We summarize principles learnt from Drosophila, where underlying sensory neuronal mechanisms are illustrated in great detail. Moreover, we compare these principles with findings in other animals, where such coordination plays prominent roles. These examples suggest that the use of gustatory information for feeding coordination has an ancient origin and is prevalent throughout the animal kingdom.

 

Publication of IMPRS-LS student Jeongyoon Choi

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Choi J, Bachmann AL, Tauscher K, Benda C, Fierz B, Müller J.
Nat Struct Mol Biol, 2017, [Epub ahead of print].
doi: 10.1038/nsmb.3488

DNA binding by PHF1 prolongs PRC2 residence time on chromatin and thereby promotes H3K27 methylation.

Polycomb repressive complex 2 (PRC2) trimethylates histone H3 at lysine 27 to mark genes for repression. We measured the dynamics of PRC2 binding on recombinant chromatin and free DNA at the single-molecule level using total internal reflection fluorescence (TIRF) microscopy. PRC2 preferentially binds free DNA with multisecond residence time and midnanomolar affinity. PHF1, a PRC2 accessory protein of the Polycomblike family, extends PRC2 residence time on DNA and chromatin. Crystallographic and functional studies reveal that Polycomblike proteins contain a winged-helix domain that binds DNA in a sequence-nonspecific fashion. DNA binding by this winged-helix domain accounts for the prolonged residence time of PHF1-PRC2 on chromatin and makes it a more efficient H3K27 methyltranferase than PRC2 alone. Together, these studies establish that interactions with DNA provide the predominant binding affinity of PRC2 for chromatin. Moreover, they reveal the molecular basis for how Polycomblike proteins stabilize PRC2 on chromatin and stimulate its activity.

 

Publication of IMPRS-LS student Elisabeth Karg

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Karg E, Smets M, Ryan J, Forné I, Qin W, Mulholland CB, Kalideris G, Imhof A, Bultmann S, Leonhardt H.
J Mol Biol, 2017, [Epub ahead of print].
doi: 10.1016/j.jmb.2017.10.014

Ubiquitome analysis reveals PCNA-associated factor 15 (PAF15) as a specific ubiquitination target of UHRF1 in embryonic stem cells.

Ubiquitination is a multifunctional posttranslational modification controlling the activity, subcellular localization and stability of proteins. The E3 ubiquitin ligase UHRF1 is an essential epigenetic factor that recognizes repressive histone marks as well as hemi-methylated DNA and recruits DNMT1. To explore enzymatic functions of UHRF1 beyond epigenetic regulation we conducted a comprehensive screen in mouse embryonic stem cells to identify novel ubiquitination targets of UHRF1 and its paralogue UHRF2. We found differentially ubiquitinated peptides associated with a variety of biological processes such as transcriptional regulation and DNA damage response. Most prominently, we identified PCNA-associated factor 15 (PAF15, also known as Pclaf, Ns5atp9, KIAA0101 and OEATC-1) as a specific ubiquitination target of UHRF1. Although the function of PAF15 ubiquitination in translesion DNA synthesis (TLS) is well characterized, the respective E3 ligase had been unknown. We could show that UHRF1 ubiquitinates PAF15 at Lys 15 and Lys 24 and promotes its binding to PCNA during late S-phase. In summary, we identified novel ubiquitination targets that link UHRF1 to transcriptional regulation and DNA damage response.