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Baek, K., Scott, D.C., and Schulman, B.A.
Curr Opin Struct Biol, 2020, 67, 101-109
doi: 10.1016/j.sbi.2020.10.007

NEDD8 and ubiquitin ligation by cullin-RING E3 ligases

RING E3s comprise the largest family of ubiquitin (UB) and ubiquitin-like protein (UBL) ligases. RING E3s typically promote UB or UBL transfer from the active site of an associated E2 enzyme to a distally-recruited substrate. Many RING E3s - including the cullin-RING ligase family - are multifunctional, interacting with various E2s (or other E3s) to target distinct proteins, transfer different UBLs, or to initially modify substrates with UB or subsequently elongate UB chains. Here we consider recent structures of cullin-RING ligases, and their partner E2 enzymes, representing ligation reactions. The studies collectively reveal multimodal mechanisms - interactions between ancillary E2 or E3 domains, post-translational modifications, or auxiliary binding partners - directing cullin-RING E3-E2 enzyme active sites to modify their specific targets.


graduationCongratulations on your PhD!


Carina Trummer
Mechanistic and functional insights into the recognition and regulation of DNA modifications by UHRF1, DNMT1 and TET proteins
RG: Heinrich Leonhardt

 


 

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Pinci, F., Gaidt, M.M., Jung, C., Kuut, G., Jackson, M.A., Bauernfried, S., and Hornung, V.
(IMPRS-LS students are in bold)
J Biol Chem, 2020, [online ahead of print].
DOI: 10.1074/jbc.RA120.015248

C-tag TNF: a reporter system to study TNF shedding

TNF is a highly pro-inflammatory cytokine that contributes not only to the regulation of immune responses but also to the development of severe inflammatory diseases. TNF is synthesized as a transmembrane protein, which is further matured via proteolytic cleavage by metalloproteases such as ADAM17, a process known as shedding. At present, TNF is mainly detected by measuring the precursor or the mature cytokine of bulk cell populations by techniques such as ELISA or immunoblotting. However, these methods do not provide information on the exact timing and extent of TNF cleavage at single-cell resolution and they do not allow the live visualization of shedding events. Here, we generated C-tag TNF as a genetically encoded reporter to study TNF shedding at the single-cell level. The functionality of the C-tag TNF reporter is based on the exposure of a cryptic epitope on the C-terminus of the transmembrane portion of pro-TNF upon cleavage. In both denatured and non-denatured samples, this epitope can be detected by a nanobody in a highly sensitive and specific manner only upon TNF shedding. As such, C-tag TNF can successfully be used for the detection of TNF cleavage in flow cytometry and live-cell imaging applications. We furthermore demonstrate its applicability in a forward genetic screen geared toward the identification of genetic regulators of TNF maturation. In summary, the C-tag TNF reporter can be employed to gain novel insights into the complex regulation of ADAM-dependent TNF shedding.


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Alankus, B., Ecker, V., Vahl, N., Braun, M., Weichert, W., Macher-Göppinger, S., Gehring, T., Neumayer, T., Zenz, T., Buchner, M., and Ruland, J.
J Exp Med, 2021, 218, [Epub ahead of print].
DOI: 10.1084/jem.20200517

Pathological RANK signaling in B cells drives autoimmunity and chronic lymphocytic leukemia

Clinical evidence suggests alterations in receptor activator of NF-κB (RANK) signaling are key contributors to B cell autoimmunity and malignancy, but the pathophysiological consequences of aberrant B cell-intrinsic RANK signaling remain unknown. We generated mice that express a human lymphoma-derived, hyperactive RANKK240E variant in B lymphocytes in vivo. Forced RANK signaling disrupted B cell tolerance and induced a fully penetrant systemic lupus erythematosus-like disease in addition to the development of chronic lymphocytic leukemia (CLL). Importantly, RANKK240E transgenic CLL cells as well as CLL cells of independent murine and of human origin depend on microenvironmental RANK ligand (RANKL) for tumor cell survival. Consequently, inhibition of the RANKL-RANK axis with anti-RANKL antibodies killed murine and human CLL cells in vitro and in vivo. These results establish pathological B cell-intrinsic RANK signaling as a potential driver of autoimmunity and B cell malignancy, and they suggest the exploitation of clinically available anti-RANKL compounds for CLL treatment.


Brenda Schulman, Ulrich Hartl and Wolfgang Baumeister receive a research grant to study Mechanisms of Parkinsons’s disease.

Brenda Schulman, Ulrich Hartl and Wolfgang Baumeister, all directors at the Max Planck Institute of Biochemistry in Martinsried, Germany have received a research grant to study the mechanisms of Parkinson’s disease from the Aligning Science Across Parkinson’s Initiative, the implementing partner of the Michael J. Fox Foundation for Parkinson’s Research is ASAP’s implementation partner. The research project will be led by Wade Harper, head of the Department of Cell Biology in the Blavatnik Institute at Havard Medical School, USA. Harper and co-investigators from the Max-Panck-Institute of Biochemistry as well as Ruben Fernandez-Busnadiego from the University of Göttingen and Judith Frydman at Stanford University, USA aim to elucidate the molecular aberrations in nerve cells that give rise to the Parkinson’s disease.

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graduationCongratulations on your PhD!


Kira Bartnik
Single-Molecule FRET Studies of Protein Function and Conformational Dynamics -
From DNA Nanotechnology to Viral and Bacterial Infections
RG: Don Lamb

 


 

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Wu, Y., Dal Maschio, M., Kubo, F., and Baier, H.
Neuron, 2020, [Epub ahead of print].
doi: 10.1016/j.neuron.2020.08.027

An Optical Illusion Pinpoints an Essential Circuit Node for Global Motion Processing

Direction-selective (DS) neurons compute the direction of motion in a visual scene. Brain-wide imaging in larval zebrafish has revealed hundreds of DS neurons scattered throughout the brain. However, the exact population that causally drives motion-dependent behaviors-e.g., compensatory eye and body movements-remains largely unknown. To identify the behaviorally relevant population of DS neurons, here we employ the motion aftereffect (MAE), which causes the well-known "waterfall illusion." Together with region-specific optogenetic manipulations and cellular-resolution functional imaging, we found that MAE-responsive neurons represent merely a fraction of the entire population of DS cells in larval zebrafish. They are spatially clustered in a nucleus in the ventral lateral pretectal area and are necessary and sufficient to steer the entire cycle of optokinetic eye movements. Thus, our illusion-based behavioral paradigm, combined with optical imaging and optogenetics, identified key circuit elements of global motion processing in the vertebrate brain.


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Tüshaus, J., Müller, S.A., Kataka, E.S., Zaucha, J., Sebastian Monasor, L., Su, M., Güner, G., Jocher, G., Tahirovic, S., Frishman, D., Simons, M., and Lichtenthaler, S.F.
(IMPRS_LS students are in bold)
EMBO J, 2020, e105693, online ahead of print.
doi: 10.15252/embj.2020105693

An optimized quantitative proteomics method establishes the cell type-resolved mouse brain secretome

To understand how cells communicate in the nervous system, it is essential to define their secretome, which is challenging for primary cells because of large cell numbers being required. Here, we miniaturized secretome analysis by developing the "high-performance secretome protein enrichment with click sugars" (hiSPECS) method. To demonstrate its broad utility, hiSPECS was used to identify the secretory response of brain slices upon LPS-induced neuroinflammation and to establish the cell type-resolved mouse brain secretome resource using primary astrocytes, microglia, neurons, and oligodendrocytes. This resource allowed mapping the cellular origin of CSF proteins and revealed that an unexpectedly high number of secreted proteins in vitro and in vivo are proteolytically cleaved membrane protein ectodomains. Two examples are neuronally secreted ADAM22 and CD200, which we identified as substrates of the Alzheimer-linked protease BACE1. hiSPECS and the brain secretome resource can be widely exploited to systematically study protein secretion and brain function and to identify cell type-specific biomarkers for CNS diseases.


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Agarwal, R., and Duderstadt, K.E.
Nat Commun, 2020, 11, 4714.
doi: 10.1038/s41467-020-18456-y

Multiplex flow magnetic tweezers reveal rare enzymatic events with single molecule precision

The application of forces and torques on the single molecule level has transformed our understanding of the dynamic properties of biomolecules, but rare intermediates have remained difficult to characterize due to limited throughput. Here, we describe a method that provides a 100-fold improvement in the throughput of force spectroscopy measurements with topological control, which enables routine imaging of 50,000 single molecules and a 100 million reaction cycles in parallel. This improvement enables detection of rare events in the life cycle of the cell. As a demonstration, we characterize the supercoiling dynamics and drug-induced DNA break intermediates of topoisomerases. To rapidly quantify distinct classes of dynamic behaviors and rare events, we developed a software platform with an automated feature classification pipeline. The method and software can be readily adapted for studies of a broad range of complex, multistep enzymatic pathways in which rare intermediates have escaped classification due to limited throughput.


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Gehrlach, D.A., Weiand, C., Gaitanos, T.N., Cho, E., Klein, A.S., Hennrich, A.A., Conzelmann, K.K., and Gogolla, N. (IMPRS-LS students are in bold)
Elife, 2020, 9.
doi: 10.7554/eLife.55585

A whole-brain connectivity map of mouse insular cortex

The insular cortex (IC) plays key roles in emotional and regulatory brain functions and is affected across psychiatric diseases. However, the brain-wide connections of the mouse IC have not been comprehensively mapped. Here, we traced the whole-brain inputs and outputs of the mouse IC across its rostro-caudal extent. We employed cell-type-specific monosynaptic rabies virus tracings to characterize afferent connections onto either excitatory or inhibitory IC neurons, and adeno-associated viral tracings to label excitatory efferent axons. While the connectivity between the IC and other cortical regions was highly bidirectional, the IC connectivity with subcortical structures was often unidirectional, revealing prominent cortical-to-subcortical or subcortical-to-cortical pathways. The posterior and medial IC exhibited resembling connectivity patterns, while the anterior IC connectivity was distinct, suggesting two major functional compartments. Our results provide insights into the anatomical architecture of the mouse IC and thus a structural basis to guide investigations into its complex functions.