Biochemical Journal最新文献

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.

Axonal transport is crucial for neuronal health and function, facilitating the delivery of newly synthesized material from the soma via anterograde transport and the removal of aged proteins and damaged organelles for degradation via retrograde transport. Emerging evidence links Parkinson's disease (PD)-causing mutations in the leucine-rich repeat kinase 2 (LRRK2) gene to dysfunctional axonal transport. Pathogenic LRRK2 mutations induce increased LRRK2 kinase activity, leading to the hyperphosphorylation of RAB proteins, which are key regulators of intracellular trafficking and transport. Here, we review the current literature on how LRRK2 affects the axonal transport of different cargoes, focusing on synaptic vesicle precursors, mitochondria, and autophagosomes. We further discuss how LRRK2 influences cytoskeletal dynamics and how it affects vesicle trafficking at the Golgi, which may indirectly contribute to its effect on axonal transport. This review summarizes our current understanding of how pathogenic LRRK2 hyperactivation disrupts axonal transport and how this may be linked to the neurodegeneration of PD.

Proteolysis-targeting chimeras (PROTACs) represent a novel and promising modality for probing biological systems, elucidating pharmacological mechanisms, and identifying potential therapeutic leads. The field has made significant strides, as demonstrated by the growing number of PROTACs advancing to clinical trials. Despite this progress, the development of PROTACs faces significant challenges, which is partially due to the heterobivalent nature of this class of molecules. PROTACs must simultaneously bind to a protein of interest and an E3 ubiquitin ligase. This means PROTACs are significantly larger and more complex than conventional small molecules. This complexity impacts their design and synthesis, requiring strategic approaches to create libraries of PROTACs with various combinations of target ligands, linkers, and E3 ligase-recruiting elements. To fully realise the potential of this innovative technology, there is a need for novel approaches to accelerate the development of PROTACs. This review focuses on three critical areas to accelerate PROTAC development: appropriate target selection, modular chemical synthesis, and high-throughput biological evaluation. By appropriate selection of target proteins for degradation, optimizing synthesis methods to handle the complexity of PROTAC molecules, and employing robust high-throughput biological assays to evaluate PROTAC activity, researchers in academia and industry have streamlined the development and increased the success rate of PROTAC-based discovery programmes.