Biology subjects

Duerr, K.

Publications and source records attributed to Duerr, K..

3 recordsLinked to original sources

bioRxiv2026

CrJAMs are transcriptional repressors of the monoterpenoid indole alkaloid pathway in Catharanthus roseus

Catharanthus roseus produces monoterpenoid indole alkaloids (MIAs) in response to the defense hormone jasmonate (JA). In Arabidopsis, JA regulates defense processes through MYC2 and JA-Associated MYC2a-like transcription factors (JAMs). Here we characterize two previously identified C. roseus JAMs, CrJAM2 and CrJAM3, as regulators of MIA biosynthesis. Functional screens using viral-induced gene silencing and transient promoter activity assays showed that CrJAMs repress the MIA pathway; silencing both CrJAM2 and CrJAM3 increased expression of MIA pathway genes and MIA levels while their individual overexpression repressed promoter activity. To assess their activity broadly across MIA regulatory and biosynthetic genes, we generated C. roseus hairy root cultures either constitutively overexpressing individual CrJAMs or CRISPR-Cas9 creating mutations in multiple CrJAMs, and profiled their expression by RNA-Seq. CrJAM2 overexpression decreased expression of most MIA biosynthetic genes relative to the GUS control, while double mutant lines showed increased basal expression of these genes relative to non-targeting controls. Notably, methyl JA treatment perturbs this equilibrium, reversing these trends at 24 hrs in both CrJAM overexpression and mutant lines relative to basal conditions. In summary, CrJAMs broadly repress MIA regulatory and biosynthetic gene expression and together with CrJAZ and CrMYC2a regulates MIA biosynthesis.

plant biology↗

bioRxiv2026

Antagonists Perturb the MC2R MRAP Complex and Reshape Receptor Conformations

Melanocortin 2 receptor (MC2R) is a G protein-coupled receptor (GPCR) for adrenocorticotropic hormone (ACTH), and its trafficking and signalling are associated with the melanocortin receptor accessory protein (MRAP). Mutations in either MC2R or MRAP disrupt this signalling and cause familial glucocorticoid deficiency. Here, we combine native mass spectrometry (MS) and hydrogen deuterium exchange mass spectrometry (HDX MS) to uncover how MRAP association and post-translational modification status shape the conformations of MC2R. Using native MS, we demonstrate that MC2R associates with MRAP or when MRAP is depleted the protein is extensively palmitoylated at the C-terminus. ACTH binding is restricted to the MC2R MRAP complex. By contrast antagonists shift the equilibrium toward MRAP-independent receptor populations. Our HDX MS analysis shows that ACTH binding induces global stabilisation of MC2R and the MRAP N terminus, consistent with reinforcement of the receptor accessory protein interface. Antagonist binding by contrast destabilises this interface and increases dynamics in transmembrane helix 2 (TM2). Notably, TM2 destabilisation is retained when the MRAP complex is depleted. Together, MRAP association and palmitoylation define distinct MC2R assemblies with ligand-dependent dynamics, suggesting new ways to influence MC2R pharmacology.

biochemistry↗

bioRxiv2022

Gluebodies improve crystal reliability and diversity through transferable nanobody mutations that introduce constitutive crystal contacts

The design of proteins that may assemble in a manner that is transferable and modular remains an enduring challenge. In particular, obtaining well-diffracting protein crystals suitable for characterizing ligands or drug candidates and understanding different protein conformations remains a bottleneck for structural studies. Using nanobodies as crystallization chaperones is one strategy to address the problem, but its reliability is uncharacterized and, in this study, we observed it to have a limited success rate. Here we show that by exploring and testing the nanobody-nanobody interfaces predominant in >200 combinations of surface mutations in multiple iterations we can engineer robust crystallization behaviour into the nanobody scaffold. Strikingly, this survey yielded multiple polymorphs, all mediated by the same interface. The resulting Gluebodies (Gbs) provide far superior resolution and reliability of diffraction and can be routinely generated for chaperone experiments. We furthermore show that Gbs cannot rescue intrinsically non-crystallizing proteins, but instead are a powerful approach to improve the packing and resolution limit of poorly diffracting crystals. The discovery of an engineered, preferred nanobody interface that arises under kinetic control - trapped here by irreversible crystallization - embodies a protein assembly strategy that could prove even more broadly useful for modular assembly trapped by other irreversible methods.

biochemistry↗