Most known probes
for activity-based protein profiling (ABPP) use
electrophilic groups that tag a single type of nucleophilic amino
acid to identify cases in which its hyper-reactivity underpins function.
Much important biochemistry derives from electrophilic enzyme cofactors,
transient intermediates, and labile regulatory modifications, but
ABPP probes for such species are underdeveloped. Here, we describe
a versatile class of probes for this less charted hemisphere of the
proteome. The use of an electron-rich hydrazine as the common chemical
modifier enables covalent targeting of multiple, pharmacologically
important classes of enzymes bearing diverse organic and inorganic
cofactors. Probe attachment occurs by both polar and radicaloid mechanisms,
can be blocked by molecules that occupy the active sites, and depends
on the proper poise of the active site for turnover. These traits
will enable the probes to be used to identify specific inhibitors
of individual members of these multiple enzyme classes, making them
uniquely versatile among known ABPP probes.
BACKGROUND AND PURPOSEUpon stimulation, neutrophils release their nuclear contents called neutrophil extracellular traps (NETs), which contain unfolded chromatin and lysosomal enzymes. NETs have been demonstrated to play a critical role in host defence, although the role of PGE 2 , a bioactive substance generated in inflammatory tissues, in the formation of NETs remains unclear.
EXPERIMENTAL APPROACHThe effects of PGE 2 , agonists and antagonists of its receptors, and modulators of the cAMP-PKA pathway on the formation of NETs were examined in vitro in isolated neutrophils and in vivo in a newly established mouse model.
KEY RESULTSPGE 2 inhibited PMA-induced NET formation in vitro through EP 2 and EP 4 Gαs-coupled receptors. Incubation with a cell-permeable cAMP analogue, dibutyryl cAMP, or various inhibitors of a cAMP-degrading enzyme, PDE, also suppressed NET formation. In the assay established here, where an agarose gel was s.c. implanted in mice and NET formation was detected on the surface of the gel, the extent of the NET formed was inhibited in agarose gels containing rolipram, a PDE4 inhibitor, and butaprost, an EP 2 receptor agonist.
CONCLUSIONS AND IMPLICATIONSPGE 2 inhibits NET formation through the production of cAMP. These findings will contribute to the development of novel treatments for NETosis-related diseases.
AbbreviationsBSA, bovine serum albumin;
Long-chain polyunsaturated fatty acids (LCPUFAs), such as docosahexaenoic acid (DHA, 22:6) and docosapentaenoic acid (DPA, 22:5), have versatile physiologic functions. Studies have suggested that DHA and DPA are beneficial for maintaining sperm quality. However, their mechanisms of action are still unclear because of the poor understanding of DHA/DPA metabolism in the testis. DHA and DPA are mainly stored as LCPUFA-containing phospholipids and support normal spermatogenesis. Long-chain acyl-conenzyme A (CoA) synthetase (ACSL) 6 is an enzyme that preferentially converts LCPUFA into LCPUFA-CoA. Here, we report that ACSL6 knockout (KO) mice display severe male infertility due to attenuated sperm numbers and function. ACSL6 is highly expressed in differentiating spermatids, and ACSL6 KO mice have reduced LCPUFA-containing phospholipids in their spermatids. Delayed sperm release and apoptosis of differentiated spermatids were observed in these mice. The results of this study indicate that ACSL6 contributes to the local accumulation of DHA- and DPA-containing phospholipids in spermatids to support normal spermatogenesis.—Shishikura, K., Kuroha, S., Matsueda, S., Iseki, H., Matsui, T., Inoue, A., Arita, M. Acyl-CoA synthetase 6 regulates long-chain polyunsaturated fatty acid composition of membrane phospholipids in spermatids and supports normal spermatogenic processes in mice.
The mechanisms leading to adrenal cortex development and steroid synthesis in humans remain poorly understood due to the paucity of model systems. Herein, we faithfully recapitulate human fetal adrenal cortex specification processes through stepwise induction of human induced pluripotent stem cells through posterior intermediate mesoderm-like and adrenal progenitor-like states to ultimately generate fetal zone adrenal cortex-like cells (FZLCs), as evidenced by histomorphological, ultrastructural, and transcriptome features and adrenocorticotropic hormone (ACTH)-independent Δ5 steroid biosynthesis. Furthermore, FZLC generation is promoted by SHH and inhibited by NOTCH, ACTIVIN and WNT signaling, and that steroid synthesis is amplified by ACTH/PKA signaling and blocked by inhibitors of delta5 steroid synthesis enzymes. Finally, NR5A1 promotes FZLC survival and steroidogenesis. Together, these findings provide a framework for understanding and reconstituting human adrenocortical development in vitro paving the way for cell-based therapies of adrenal insufficiency.
Chemical platforms that facilitate both the identification and elucidation of new areas for therapeutic development are necessary but lacking. Activity-based protein profiling (ABPP) leverages active site-directed chemical probes as target discovery tools that resolve activity from expression and immediately marry the targets identified with lead compounds for drug design. However, this approach has traditionally focused on predictable and intrinsic enzyme functionality. Here, we applied our activity-based proteomics discovery platform to map non-encoded and post-translationally acquired enzyme functionalities (e.g. cofactors) in vivo using chemical probes that exploit the nucleophilic hydrazine pharmacophores found in a classic antidepressant drug (e.g. phenelzine, Nardil ®). We show the probes are in vivo active and can map proteome-wide tissue-specific target engagement of the drug. In addition to engaging targets (flavoenzymes monoamine oxidase A/B) that are associated with the known therapeutic mechanism as well as several other members of the flavoenzyme family, the probes captured the previously discovered N-terminal glyoxylyl (Glox) group of Secernin-3 (SCRN3) in vivo through a divergent mechanism, indicating this functional feature has biochemical activity in the brain. SCRN3 protein is ubiquitously expressed in the brain, yet gene expression is regulated by inflammatory stimuli. In an inflammatory pain mouse model, behavioral assessment of nociception showed Scrn3 male knockout mice selectively exhibited impaired thermal nociceptive sensitivity. Our study provides a guided workflow to entangle molecular (off)targets and pharmacological mechanisms for therapeutic development.
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