Biochemical Journal最新文献

Apoptotic caspases exist not as static structures but as dynamic ensembles in solution, finely tuned by post-translational modifications and oligomerization. The fine-tuning of this ensemble by cellular cues allows caspases to influence not only apoptotic pathways but also the non-apoptotic pathways in which they are involved. These ensembles span a complex conformational landscape from well-characterized low-energy states captured in structural databases to transient high-energy intermediates that remain elusive and poorly understood. This limited structural view poses a major barrier to fully understanding how caspase activity is regulated and diversified across cellular contexts. To address this, we integrate evolutionary, folding, and mutational data with molecular dynamics simulations and network analysis to uncover a highly conserved residue network in structural space that has been faithfully passed on in sequence space over 500 million years of vertebrate evolution. This network encodes a high-energy intermediate consistently present in the ensemble of all present-day vertebrate apoptotic caspases. It not only guides folding but also scaffolds dynamic motions, functioning like a structural backbone that supports the ensemble. Building on this foundation, we identify differentially evolving networks surrounding the conserved core in initiator and effector caspase subfamilies. These variations provide thermodynamic insight into how initiators stabilize monomeric conformations while effectors favor dimeric states, revealing how evolution shapes ensembles to diversify function in protein families. Additionally, we discover conserved hub residues near an allosteric hotspot, distinct from the core network, that regulate the dynamics of surrounding evolving networks and act as control centers that modulate the conformational equilibrium within the apoptotic caspase ensemble.

In Leishmania parasites, as for their hosts, the ubiquitin (Ub) proteasome system is important for cell viability. As part of a systematic gene deletion study, it was discovered that four cysteine protease-type deubiquitinases (DUBs) are essential for parasite survival in the promastigote stage, including DUB16. Here, we have purified and characterised recombinant DUB16 from Leishmania donovani, which belongs to the Ub C-terminal hydrolase (UCH) family. DUB16 efficiently hydrolyses C-terminal aminocoumarin and rhodamine conjugates of Ub consistent with proposed cellular roles of UCH-type DUBs in regenerating free monomeric Ub from small molecule Ub adducts arising from adventitious metabolic processes. The crystal structure of DUB16 reveals a typical UCH-type DUB fold, and a relatively short and disordered cross-over loop that appears to restrict access to the catalytic cysteine. At close to stoichiometric enzyme to substrate ratios, DUB16 exhibits DUB activity towards diubiquitins linked through isopeptide bonds between Lys11, Lys48 or Lys63 residues of the proximal Ub and the C-terminus of the distal Ub. With 100-1000-fold higher turnover rates, DUB16 cleaves the ubiquitin-ribosomal L40 fusion protein to give the mature products. A DUB-targeting cysteine-reactive cyanopyrrolidine compound, IMP-1710, inhibits DUB16 activity. IMP-1710 was shown in promastigote cell viability assays to have parasite killing activity with EC50 values of 1-2 μM, comparable with the anti-leishmanial drug, miltefosine. L. mexicana parasites engineered to overproduce DUB16 showed a modest increase in resistance to IMP-1710, providing evidence that IMP-1710 inhibits DUB16 in vivo. While it is highly likely that IMP-1710 has additional targets, these results suggest that DUB16 is a potential target for the development of new anti-leishmanial compounds.