Biochemical Journal最新文献

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.

We aimed to create a high-throughput fluorimetric assay for the activity of CYP4A11, the major 20-HETE-producing enzyme. To this end, we probed 3-(6-methoxynaphthalen-2-yl)acrylic acid (MONACRA) as a potential CYP4A11 substrate. We studied its metabolism using human liver microsomes (HLM) and recombinant P450 enzymes. O-demethylation of MONACRA by cytochromes P450 creates 3-(6-hydroxynaphthalen-2-yl)acrylic acid. The bright fluorescence of the product and its clear spectral resolution from the substrate allowed us to create a fluorimetric assay of MONACRA metabolism. We tested 16 recombinant human P450 enzymes and found noticeable demethylation activity only with CYP4A11 and CYP1A2. The KM for CYP4A11 is 189±37 μM, and the kcat accounts for 67±18 min-1. CYP1A2 exhibits a KM of 161±34 μM, with a kcat value of 44±6 min-1, although this enzyme also exhibited a decreased rate of turnover at high substrate concentrations, evidencing substrate inhibition with Ksi=650±200 μM. The studies with fluvoxamine and epalrestat, specific inhibitors of CYP1A2 and CYP4A11, respectively, showed that despite the activity of recombinant CYP1A2 with MONACRA, it does not take part in its metabolism in HLM. Thus, MONACRA can be utilized as a specific fluorogenic substrate of CYP4A11. We developed a robust and sensitive automated fluorimetric assay of MONACRA demethylation and used it to compare the substrate saturation profiles in seven pooled HLM preparations with the known composition of the P450 pool. These studies demonstrated a close correlation between the rate of the main kinetic phase of MONACRA metabolism and the fractional content of CYP4A11 in the P450 pool.