Biology subjects

Schaefer, J. V.

Publications and source records attributed to Schaefer, J. V..

6 recordsLinked to original sources

bioRxiv2024

Selective targeting of TBXT with DARPins identifies regulatory networks and therapeutic vulnerabilities in chordoma

Aberrant expression of the embryonal transcription factor TBXT (also known as brachyury) drives chordoma, a rare spinal neoplasm with no effective drug therapies. The gene network regulated by TBXT is poorly understood, and strategies to disrupt its abnormal activity for therapeutic purposes are lacking. Here, we developed TBXT-targeted designed ankyrin repeat proteins (T-DARPins) that selectively bind TBXT, inhibiting its binding to DNA and expression. In chordoma cells, T-DARPins reduced cell cycle progression, spheroid formation, and tumor growth in mice and induced morphologic changes indicative of senescence and differentiation. Combining T-DARPin-mediated TBXT inhibition with transcriptomic and proteomic analyses, we determined the TBXT regulome in chordoma cells, which comprises in particular networks involved in cell cycle regulation, DNA replication and repair, embryonal cell identity, metabolic processes, and interferon response. The analysis of selected TBXT regulome components provided new insights into chordoma biology, such as the strong upregulation of IGFBP3 upon TBXT inhibition to fine-tune part of TBXTs downstream effectors. Finally, we assigned each TBXT regulome member a druggability status to create a resource for future translational studies and found high interferon response signaling in chordoma cell lines and patient tumors, which was promoted by TBXT and associated with strong sensitivity to clinically approved JAK2 inhibitors. These findings demonstrate the potential of DARPins to investigate the function of nuclear proteins to understand the regulatory networks of cancers driven by aberrant transcription factor activity, including novel entry points for targeted therapies that warrant testing in patients.

cancer biology↗

bioRxiv2022

Diversity-oriented synthesis encoded by deoxyoligonucleotides

Diversity-oriented synthesis (DOS)is a powerful strategy to prepare molecules with underrepresented features in commercial screening collections, resulting in the elucidation of novel biological mechanisms. In parallel to the development of DOS, DNA-encoded libraries (DELs) have emerged as an effective, efficient screening strategy to identify protein binders. Despite recent advancements in this field, most DEL syntheses are limited by the presence of sensitive DNA-based constructs. Here, we describe the design, synthesis, and validation experiments performed for a 3.7 million-member DEL, generated using diverse skeleton architectures with varying exit vectors, derived from DOS, to achieve structural diversity beyond what is possible by varying appendages alone. We will make this DEL available to the academic scientific community to increase access to novel structural features and accelerate early-phase drug discovery.

biochemistry↗

bioRxiv2022

Nuclear myosin VI maintains replication fork stability

The actin cytoskeleton is of fundamental importance for cellular structure and plasticity. However, abundance and function of filamentous (F-) actin in the nucleus are still controversial. Here we show that the actin-based molecular motor myosin VI contributes to the stabilization of stalled or reversed replication forks. In response to DNA replication stress, myosin VI associates with stalled replication intermediates and cooperates with the AAA ATPase WRNIP1 in protecting these structures from DNA2- mediated nucleolytic attack. Using nuclear localization sequence (NLS) and ubiquitin E3-fusion DARPins to manipulate myosin VI levels in a compartment-specific manner, we provide evidence for the direct involvement of myosin VI in the nucleus and against a contribution of the abundant cytoplasmic pool during the replication stress response.

cell biology↗

bioRxiv2021

Structural basis of adenylyl cyclase 9 activation

Adenylyl cyclase 9 (AC9) is a membrane-bound enzyme that converts ATP into cAMP. The enzyme is weakly activated by forskolin, fully activated by the G protein Gs subunit and is autoinhibited by the AC9 C-terminus. Although our recent structural studies of the AC9-Gs complex provided the framework for understanding AC9 autoinhibition, the conformational changes that AC9 undergoes in response to activator binding remains poorly understood. Here, we present the cryo-EM structures of AC9 in several distinct states: (i) AC9 bound to a nucleotide inhibitor MANT-GTP, (ii) bound to an artificial activator (DARPin C4) and MANT-GTP, (iii) bound to DARPin C4 and a nucleotide analogue ATPS, (iv) bound to Gs and MANT-GTP. The artificial activator DARPin C4 partially activates AC9 by binding at a site that overlaps with the Gs binding site. Together with the previously observed occluded and forskolin-bound conformations, structural comparisons of AC9 in the four new conformations show that secondary structure rearrangements in the region surrounding the forskolin binding site are essential for AC9 activation. One Sentence SummaryCryo-EM reveals activator-induced conformational changes in adenylyl cyclase AC9

biochemistry↗

bioRxiv2020

Asymmetric requirement of Dpp/BMP morphogen dispersal in the Drosophila wing disc

How morphogen gradients control patterning and growth in developing tissues remains largely unknown due to lack of tools manipulating morphogen gradients. Here, we generate two membrane-tethered protein binders that manipulate different aspects of Decapentaplegic (Dpp), a morphogen required for overall patterning and growth of the Drosophila wing. "HA trap" is based on a single-chain variable fragment (scFv) against the HA tag that traps HA-Dpp to mainly block its dispersal, while "Dpp trap" is based on a Designed Ankyrin Repeat Protein (DARPin) against Dpp that traps Dpp to block both its dispersal and signaling. Using these tools, we found that, while posterior patterning and growth require Dpp dispersal, anterior patterning and growth largely proceed without Dpp dispersal. We show that dpp transcriptional refinement from an initially uniform to a localized expression and persistent signaling in transient dpp source cells render the anterior compartment robust against the absence of Dpp dispersal. Furthermore, despite a critical requirement of dpp for the overall wing growth, neither Dpp dispersal nor direct signaling is critical for lateral wing growth after wing pouch specification. These results challenge the long-standing dogma that Dpp dispersal is strictly required to control and coordinate overall wing patterning and growth.

developmental biology↗

bioRxiv2018

DARPins recognizing mTFP1 as novel reagents for in vitro and in vivo protein manipulations

Over the last few years, protein-based affinity reagents have proven very helpful in cell and developmental biology. While many of these versatile small proteins can be expressed both in the intracellular and extracellular milieu in cultured cells and in living organisms, they can also be functionalized by fusing them to different protein domains in order to regulate or modulate their target proteins in diverse manners. For example, protein binders have been employed to degrade, trap, localize or enzymatically modify specific target proteins. Whereas binders to many endogenous proteins or small protein tags have been generated, also several affinity reagents against fluorescent proteins have been created and used to manipulate target proteins tagged with the corresponding fluorescent protein. Both of these approaches have resulted in improved methods for cell biological and developmental studies. While binders against GFP and mCherry have been previously isolated and validated, we now report the generation and utilization of designed ankyrin repeat proteins (DARPins) against the monomeric teal fluorescent protein 1 (mTFP1). Here we use the generated DARPins to delocalize Rab proteins to the nuclear compartment, in which they cannot fulfill their regular functions anymore. In the future, such manipulations might enable the production of acute loss-of-function phenotypes in different cell types or living organisms based on direct protein manipulation rather than on genetic loss-of-function analyses.\n\nSummary statementStructural characterization of two novel DARPins (designed ankyrin repeat proteins) recognizing the monomeric teal fluorescent protein 1 (mTFP1) and their functionalization for protein manipulation strategies in cultured cells and potentially in living organisms.

cell biology↗