Biology subjects

Kunzelmann, S.

Publications and source records attributed to Kunzelmann, S..

9 recordsLinked to original sources

bioRxiv2026

BacPROTACs outperform inhibitors in Mycobacterium tuberculosis

Antibiotic discovery has long relied on occupancy-driven inhibition, leaving a vast number of potential bacterial targets undrugged.1 Targeted protein degradation offers a mechanistically distinct alternative to inhibition, yet its application to antibacterial drug discovery remains largely unexplored.2-4 Here we describe the development of first-in-class heterobifunctional bacterial proteolysis targeting chimeras (BacPROTACs) directed against an essential Mycobacterium tuberculosis protein, 4-phosphopantetheinyl transferase (PptT).5 Leveraging the modular architecture of BacPROTACs, we repurposed PptT inhibitors by incorporating them into degraders, yielding compounds with markedly improved antimycobacterial activity. Integrating in vitro and cellular approaches, we developed a characterisation pipeline to assess protein degradation in bacteria, applicable to future BacPROTAC programmes. Our study establishes targeted protein degradation as a strategy for antibacterial drug discovery.

microbiology↗

bioRxiv2026

Phosphoregulated SMCR8-FIP200 interaction connects the ALS/FTD-linked C9orf72 complex to autophagy initiation and mitochondrial quality control

Hexanucleotide (GGGGCC) repeat expansions in the non-coding region of C9ORF72 are a major genetic cause of ALS/FTD and reduce C9orf72-SMCR8-WDR41 complex levels, but how this contributes to autophagy-lysosome dysfunction and previously reported mitochondrial quality-control defects in C9ORF72-ALS/FTD remains unclear. Here we identify a direct interaction between SMCR8 and the FIP200 subunit of the ULK1/2 autophagy initiation complex, mediated by two FIP200-interacting region (FIR) motifs in a disordered SMCR8 loop. Phosphorylation of these motifs by ULK1/2 or TBK1 strengthens binding and promotes ULK1/2 complex association in cells. Stabilising the SMCR8-FIP200 interaction suppresses Parkin-dependent mitophagy, whereas both stabilisation and weakening impair deferiprone-induced mitophagy, while leaving bulk autophagy, lysophagy and ivermectin-induced mitophagy largely intact. These findings define a regulated C9orf72-ULK1/2 axis and provide a mechanistic framework by which repeat-expansion-associated reduction in C9orf72 complex abundance may contribute to previously observed mitochondrial quality-control defects in C9ORF72-ALS/FTD.

cell biology↗

bioRxiv2025

A structural and regulatory framework for Atg9-containing vesicle formation and their Atg1-dependent remodelling during autophagy initiation

Autophagy is a complex intracellular degradation pathway that depends on the coordinated interplay between the core autophagy machinery and diverse membrane sources to drive the de novo formation of double-membrane vesicles, known as autophagosomes. Golgi-derived Atg9-containing vesicles are essential for this process, delivering membranes to the pre-autophagosomal structure (PAS). These vesicles contain the transmembrane proteins Atg9 and Atg27 and the peripheral membrane protein Atg23; however, the nature, function, and regulation of their interactions remain poorly understood. Here, we systematically dissect the molecular interactions between Atg9, Atg23 and Atg27, and uncover their regulation in space and time. The bipartite binding mode by which Atg23 engages Atg9 provides a structural model for how Atg23 promotes vesicle budding. Furthermore, Atg1-dependent phosphorylation of Atg9 remodels its interactions with Atg23 and Atg27 at the PAS to support autophagy initiation. Together, these findings establish a molecular and regulatory framework for the earliest steps of autophagy.

biochemistry↗

bioRxiv2025

Structural Basis of CSN-mediated SCF Deneddylation

Cullin-RING ligases (CRLs) are the largest family of E3 ligases, with their ubiquitination activity dynamically regulated by neddylation and deneddylation by the COP9 signalosome (CSN). CSN-mediated deneddylation not only deactivates CRLs but also enables substrate receptor exchange, fine-tuning CRL specificity. CSN has emerged as a promising drug target, yet the structural basis underlying its catalytic mechanism remains insufficiently understood. To address this, we used cryo-electron microscopy (cryo-EM) analysis of CSN-CRL1 (SCF) complexes to uncover distinct functional states, capturing key intermediates of the deneddylation cycle. The earliest state represents an initial docking step in which CSN remains autoinhibited. In contrast, the catalytic intermediate reveals the fully engaged state, with the CSN5 Ins-1 loop, RBX1 RING, and neddylated Cullin WHB domains repositioned for isopeptide bond cleavage. We further resolve four dissociation intermediates that define the stepwise release of CSN from its deneddylated product, highlighting a central role for RBX1RING in stabilising key interactions throughout this process. Additionally, our structures reveal the previously uncharacterised position of CSNAP, which integrates into the CSN scaffold at a groove formed by CSN3 and CSN8. Together, our study provides a comprehensive mechanistic model of CSN function, linking CRL recognition, catalytic activation, and stepwise disengagement. These insights lay the groundwork for the rational design of CSN inhibitors, offering new opportunities to modulate CRL activity for therapeutic applications. Key TakeawaysO_LIHigh-resolution cryo-EM structures capture key intermediates of the CSN-mediated SCF deneddylation cycle C_LIO_LIStructural identification of CSNAP places it within a previously uncharacterized groove at the interface of CSN3 and CSN8. C_LIO_LIPre-catalytic and catalytic states define the conformational transitions of CSN5Ins-1, RBX1RING, and N8WHB that enable isopeptide bond cleavage. C_LIO_LIFour dissociation intermediates reveal a sequential pathway for CSN disengagement, with RBX1RING playing a central role in stabilising key interactions. C_LI

biochemistry↗

bioRxiv2024

Structural and functional characterization of the KHNYN extended-diKH domain for mediating ZAP antiviral activity

Zinc finger antiviral protein (ZAP) binds CpG dinucleotides in viral RNA and targets them for decay. ZAP interacts with several cofactors to form the ZAP antiviral system, including KHNYN, a multidomain endoribonuclease required for ZAP-mediated RNA decay. However, it is unclear how the individual domains in KHNYN contribute to its activity. Here, we demonstrate that the KHNYN amino terminal extended-diKH (ex-diKH) domain is required for antiviral activity and present its crystal structure. The structure belongs to a rare group of KH-containing domains, characterized by a non-canonical arrangement between two type-1 KH modules, with an additional helical bundle. N4BP1 is a KHNYN paralog with an ex-diKH domain that functionally complements the KHNYN ex-diKH domain. Interestingly, the ex-diKH domain structure is present in N4BP1-like proteins in lancelets, which are basal chordates, indicating that it is evolutionarily ancient. While many KH domains demonstrate RNA binding activity, biolayer interferometry and electrophoretic mobility shift assays indicate that the KHNYN ex-diKH domain does not bind RNA. Furthermore, residues required for canonical KH domains to bind RNA are not required for KHNYN antiviral activity. By contrast, an inter-KH domain cleft in KHNYN is a potential protein-protein interaction site and mutations that eliminate arginine salt bridges at the edge of this cleft decrease KHNYN antiviral activity. This suggests that this domain could be a binding site for an unknown KHNYN cofactor.

microbiology↗

bioRxiv2024

Activation loop phosphorylation and cGMP saturation of PKG regulate egress of malaria parasites.

The cGMP-dependent protein kinase (PKG) is the sole cGMP sensor in malaria parasites, acting as an essential signalling hub to govern key developmental processes throughout the parasite life cycle. Despite the importance of PKG in the clinically relevant asexual blood stages, many aspects of malarial PKG regulation, including the importance of phosphorylation, remain poorly understood. Here we use genetic and biochemical approaches to show that reduced cGMP binding to cyclic nucleotide binding domain B does not affect in vitro kinase activity but prevents parasite egress. Similarly, we show that phosphorylation of a key threonine residue (T695) in the activation loop is dispensable for kinase activity in vitro but is essential for in vivo PKG function, with loss of T695 phosphorylation leading to aberrant phosphorylation events across the parasite proteome and changes to the substrate specificity of PKG. Our findings indicate that Plasmodium PKG is uniquely regulated to transduce signals crucial for malaria parasite development.

microbiology↗

bioRxiv2021

N4BP1 is dimerization-dependent linear ubiquitin reader regulating TNFR1 signalling through linear ubiquitin binding and Caspase-8-mediated processing

Signalling through TNFR1 modulates proinflammatory gene transcription and programmed cell death, and its impairment causes autoimmune diseases and cancer. NEDD4-binding protein 1 (N4BP1) was recently identified as a critical suppressor of proinflammatory cytokine production1, whose mode of action remained unknown. Here, we show that N4BP1 is a novel linear ubiquitin reader that negatively regulates NF{kappa}B signalling by its unique dimerizationdependent ubiquitin-binding module that we named LUBIN. Dimeric N4BP1 strategically positions two non-selective ubiquitin-binding domains to ensure exclusive recognition of linear ubiquitin. Under proinflammatory conditions, N4BP1 is recruited to the nascent TNFR1 signalling complex, where it regulates duration of proinflammatory signalling in LUBIN-dependent manner. N4BP1 deficiency accelerates TNF-induced cell death by increasing complex II assembly. Under proapoptotic conditions, Caspase-8 mediates proteolytic processing of N4BP1 and the resulting cleavage fragment of N4BP1, which retains the ability to bind linear ubiquitin, is rapidly degraded by the 26S proteasome, accelerating apoptosis. In summary, our findings demonstrate that N4BP1 dimerization creates a unique linear ubiquitin reader that ensures timely and coordinated regulation of TNFR1-mediated inflammation and cell death.

biochemistry↗

bioRxiv2019

The speed of GTP hydrolysis determines GTP cap size and controls microtubule stability

Microtubules are bistable cytoskeletal polymers whose function depends on their property to switch between states of growth and shrinkage 1. Growing microtubules are thought to be stabilized by a GTP cap at their ends 2-5. The nature of this cap, however, is still poorly understood. How GTP hydrolysis determines the properties of the GTP cap and hence microtubule stability is unclear. End Binding proteins (EBs) recruit a diverse range of regulators of microtubule function to growing microtubule ends 6. Whether these regulatory platforms at growing microtubule ends are identical to the GTP cap is not known. Using mutated human tubulin with blocked GTP hydrolysis, we demonstrate in microscopy-based in vitro reconstitutions that EB proteins bind with high affinity to the GTP conformation of microtubules. Slowing-down GTP hydrolysis leads to extended GTP caps and consequently hyper-stable microtubules. Single molecule experiments reveal that the microtubule conformation gradually changes in the cap as GTP is hydrolyzed. These results demonstrate the critical importance of the kinetics of GTP hydrolysis for microtubule stability; and establish that the GTP cap coincides with the EB-binding regulatory hub that modulates microtubule cytoskeleton function in cells.

biochemistry↗

bioRxiv2019

Protein ubiquitylation is essential for the schizont to merozoite transition in Plasmodium falciparum blood-stage development

Ubiquitylation is a common post translational modification of eukaryotic proteins and in the human malaria parasite, Plasmodium falciparum (Pf) overall ubiquitylation increases in the transition from intracellular schizont to extracellular merozoite stages in the asexual blood stage cycle. Here, we identify specific ubiquitylation sites of protein substrates in three intracellular parasite stages and extracellular merozoites; a total of 1464 sites in 546 proteins were identified (data available via ProteomeXchange with identifier PXD014998). 469 ubiquitylated proteins were identified in merozoites compared with only 160 in the preceding intracellular schizont stage, indicating a large increase in protein ubiquitylation associated with merozoite maturation. Following merozoite invasion of erythrocytes, few ubiquitylated proteins were detected in the first intracellular ring stage but as parasites matured through trophozoite to schizont stages the extent of ubiquitylation increased. We identified commonly used ubiquitylation motifs and groups of ubiquitylated proteins in specific areas of cellular function, for example merozoite pellicle proteins involved in erythrocyte invasion, exported proteins, and histones. To investigate the importance of ubiquitylation we screened ubiquitin pathway inhibitors in a parasite growth assay and identified the ubiquitin activating enzyme (UBA1 or E1) inhibitor MLN7243 (TAK-243) to be particularly effective. This small molecule was shown to be a potent inhibitor of recombinant PfUBA1, and a structural homology model of MLN7243 bound to the parasite enzyme highlights avenues for the development of P. falciparum specific inhibitors. We created a genetically modified parasite with a rapamycin-inducible functional deletion of uba1; addition of either MLN7243 or rapamycin to the recombinant parasite line resulted in the same phenotype, with parasite development blocked at the late schizont stage. These results indicate that the intracellular target of MLN7243 is UBA1, and this activity is essential for the final differentiation of schizonts to merozoites. The ubiquitylation of many merozoite proteins and their disappearance in ring stages are consistent with the idea that ubiquitylation leads to their destruction via the proteasome once their function is complete following invasion, which would allow amino acid recycling in the period prior to the parasites elaboration of a new food vacuole.

microbiology↗