Biology subjects

Hinterndorfer, M.

Publications and source records attributed to Hinterndorfer, M..

6 recordsLinked to original sources

bioRxiv2024

A multivalent adaptor mechanism drives the nuclear import of proteasomes

Nuclear protein homeostasis, including the turnover of transcription factors, critically depends on nuclear proteasomes. After each cell division, proteasomes need to be re-imported into the newly formed nucleus in a highly dynamic process that requires the largely unstructured protein AKIRIN2. However, how AKIRIN2 orchestrates this process and, more generally, how large protein complexes are translocated into the nucleus remains poorly understood. Here, we have used an integrated approach combining protein-wide saturation mutagenesis screens, cryoEM, and biochemical reconstitution to characterize AKIRIN2 as a scaffold protein that coordinates the stepwise assembly of an importin cluster around the proteasome. Through surveying every possible single amino acid substitution in AKIRIN2 using FACS- and microscopy-based genetic screens, we establish a comprehensive map of functionally relevant residues and binding interfaces in structured and disordered protein regions. Integrating these results with cryoEM analysis reveals a wing helix in a disordered region of AKIRIN2 that plays a crucial role in stabilizing proteasome interactions. Upon primary binding, AKIRIN2 homodimers recruit the importin IPO9, which in turn facilitates the binding of a second AKIRIN2 homodimer that recruits additional importins. Together, this multivalent molecular assembly amplifies the number of nuclear localisation signals and, thereby, triggers efficient proteasome translocation into the nucleus. Inside the nucleus, RanGTP rapidly dissociates importins, and AKIRIN2 is degraded by the proteasome in a ubiquitin-independent manner. Beyond mechanistically resolving the nuclear import of proteasomes, we propose that multivalent adaptor proteins like AKIRIN2 orchestrate the import of other macromolecular complexes and thereby dynamically control the composition of the nuclear proteome.

molecular biology↗

bioRxiv2024

High-throughput diversification of protein-ligand surfaces to discover chemical inducers of proximity

Chemical inducers of proximity (CIPs) stabilize biomolecular interactions, often causing an emergent rewiring of cellular biochemistry1,2. While rational design strategies can expedite the discovery of heterobifunctional CIPs, monovalent, molecular glue-like CIPs have relied predominantly on serendipity3. Envisioning a prospective approach to discover molecular glues for a pre-selected target, we hypothesized that pre-existing ligands could be systematically decorated with chemical modifications to empirically discover protein-ligand surfaces that are tuned to cooperatively engage another protein interface. Here, we used sulfur(VI)-fluoride exchange (SuFEx)-based high-throughput chemistry (HTC) to install 3,163 structurally diverse chemical building blocks onto ENL and BRD4 ligands and then screened the crude products for degrader activity. This revealed dHTC1, a potent, selective, and stereochemistry-dependent degrader of ENL. It recruits CRL4CRBN to ENL through an extended interface of protein-protein and protein-ligand contacts, but only after pre-forming the ENL:dHTC1 complex. We also characterized two structurally distinct BRD4 degraders, including dHTC3, a molecular glue that selectively dimerizes the first bromodomain of BRD4 to SCFFBXO3, an E3 ligase not previously accessible for chemical rewiring. Altogether, this study introduces HTC as a facile tool to discover new CIPs and actionable cellular effectors of proximity pharmacology.

biochemistry↗

bioRxiv2024

Inhibitor-induced supercharging of kinase turnover via endogenous proteolytic circuits

Targeted protein degradation has emerged as a promising new pharmacological strategy. Traditionally, it relies on small molecules that induce proximity between a target protein and an E3 ubiquitin ligase to prompt target ubiquitination and degradation by the proteasome. Sporadic reports indicated that ligands designed to inhibit a target can also induce its destabilization. Among others, this has repeatedly been observed for kinase inhibitors. However, we lack an understanding of the frequency, generalizability, and mechanistic underpinnings of these phenomena. To address this knowledge gap, we generated dynamic abundance profiles of 98 kinases after cellular perturbations with 1570 kinase inhibitors, revealing 160 selective instances of inhibitor-induced kinase destabilization. Kinases prone to degradation are frequently annotated as HSP90 clients, thus affirming chaperone deprivation as an important route of destabilization. However, detailed investigation of inhibitor-induced degradation of LYN, BLK and RIPK2 revealed a differentiated, common mechanistic logic where inhibitors function by inducing a kinase state that is more efficiently cleared by endogenous degradation mechanisms. Mechanistically, effects can manifest by ligand-induced changes in cellular activity, localization, or multimerization which may be triggered by direct target engagement or network effects. Collectively, our data suggest that inhibitor-induced kinase degradation is a common event and positions supercharging of endogenous degradation circuits as an alternative to classical proximity-inducing degraders.

molecular biology↗

bioRxiv2023

An intramolecular bivalent degrader glues an intrinsic BRD4-DCAF16 interaction

Targeted protein degradation is a pharmacological modality based on the induced proximity of an E3 ubiquitin ligase and a target protein to promote target ubiquitination and proteasomal degradation. This has been achieved either via bifunctional compounds (PROTACs) composed of two separate warheads that individually bind the target and E3 ligase, or via molecular glues that monovalently bind either the ligase or the target1-4. Using orthogonal genetic screening, biophysical characterization, and structural reconstitution, we investigate the mode of action of bifunctional BRD2/4 degraders (IBG1-4) and find that - instead of connecting target and ligase in trans as PROTACs do - they simultaneously engage two adjacent domains of the target protein in cis. This conformational change glues BRD4 to the E3 ligases DCAF11 or DCAF16, leveraging intrinsic target-ligase affinities which, albeit pre-existing, do not translate to BRD4 degradation in absence of compound. Structural insights into the ternary BRD4:IBG1:DCAF16 complex guided the rational design of improved degraders of low picomolar potency. We thus introduce a new modality in targeted protein degradation, termed intramolecular bivalent glues (IBGs), which work by bridging protein domains to enhance surface complementarity with E3 ligases for productive ubiquitination and degradation.

biochemistry↗

bioRxiv2022

Precision RNAi using synthetic shRNAmir target sites

Loss-of-function genetic tools are widely applied for validating therapeutic targets, but their utility remains limited by incomplete on- and uncontrolled off-target effects. We describe artificial RNA interference (ARTi) based on synthetic, ultra-potent, off-target-free shRNAs that enable efficient and inducible suppression of any gene upon introduction of a synthetic target sequence into non-coding transcript regions. ARTi establishes a scalable loss-of-function tool with full control over on- and off-target effects.

genetics↗

bioRxiv2022

SPOP targets the immune transcription factor IRF1 for proteasomal degradation

Adaptation of the functional proteome is essential to counter pathogens during infection, yet precisely timed degradation of these response proteins after pathogen clearance is likewise key to preventing autoimmunity. Interferon Regulatory Factor 1 (IRF1) plays an essential role as a transcription factor in driving the expression of immune response genes during infection. The striking difference in functional output with other IRFs, is that IRF1 also drives the expression of various cell cycle inhibiting factors, making it an important tumor suppressor. Thus, it is critical to regulate the abundance of IRF1 to achieve a "Goldilocks" zone in which there is sufficient IRF1 to prevent tumorigenesis, yet not too much which could drive excessive immune activation. Using genetic screening, we identified the E3 ligase receptor Speckle Type BTB/POZ Protein (SPOP) to mediate IRF1 proteasomal turnover. We identified four S/T-rich degrons in IRF1 required for its SPOP MATH domain-dependent turnover. In the absence of SPOP, increased IRF1 protein levels functionally drive increased transcription of IRF1-response-genes, underpinning the biological significance of SPOP in curtailing IRF1 protein abundance.

cell biology↗