Chemoproteomic strategy identified p120-catenin glutathionylation regulates E-cadherin degradation and cell migration.

Identification of cysteines with high oxidation susceptibility is important for understanding redox-mediated biological processes. In this report, we report a chemical proteomic strategy that finds cysteines with high susceptibility to S-glutathionylation. Our proteomic strategy, named clickable glutathione-based isotope-coded affinity tag (G-ICAT), identified 1,518 glutathionylated cysteines while determining their relative levels of glutathionylated and reduced forms upon adding hydrogen peroxide. Among identified cysteines, we demonstrated that CTNND1 (p120) C692 has high susceptibility to glutathionylation. Also, p120 wild type (WT), compared to C692S, induces its dissociation from E-cadherin under oxidative stress, such as glucose depletion. p120 and E-cadherin dissociation correlated with E-cadherin destabilization via its proteasomal degradation. Lastly, we showed that p120 WT, compared to C692S, increases migration and invasion of MCF7 cells under glucose depletion, supporting a model that p120 C692 glutathionylation increases cell migration and invasion by destabilization of E-cadherin, a core player in cell-cell adhesion.

Long chain acyl-CoA synthetase 6 facilitates the local distribution of di-docosahexaenoic acid- and ultra-long-chain-PUFA-containing phospholipids in the retina to support normal visual function in mice.

Docosahexaenoic acid (DHA) and ultra-long-chain polyunsaturated fatty acids (ULC-PUFAs) are uniquely enriched in membrane phospholipids of retinal photoreceptors. Several studies have shown that di-DHA- and ULC-PUFA-containing phospholipids in photoreceptors have an important role in maintaining normal visual function; however, the molecular mechanisms underlying the synthesis and enrichment of these unique lipids in the retina, and their specific roles in retinal function remain unclear. Long-chain acyl-coenzyme A (CoA) synthetase 6 (ACSL6) preferentially converts DHA into DHA-CoA, which is a substrate during DHA-containing lipid biosynthesis. Here, we report that Acsl6 mRNA is expressed in the inner segment of photoreceptor cells and the retinal pigment epithelial cells, and genetic deletion of ACSL6 resulted in the selective depletion of di-DHA- and ULC-PUFA-containing phospholipids, but not mono-DHA-containing phospholipids in the retina. MALDI mass spectrometry imaging (MALDI-MSI) revealed the selective distribution of di-DHA- and ULC-PUFA-containing phospholipids in the photoreceptor outer segment (OS). Electroretinogram of Acsl6-/- mice exhibited photoreceptor cell-derived visual impairment, whereas the expression levels and localization of opsin proteins were unchanged. Acsl6-/- mice exhibited an age-dependent progressive decrease of the thickness of the outer nuclear layers, whereas the inner nuclear layers and OSs were normal. These results demonstrate that ACSL6 facilitates the local enrichment of di-DHA- and ULC-PUFA-containing phospholipids in the retina, which supports normal visual function and retinal homeostasis.

Phenelzine-based probes reveal Secernin-3 is involved in thermal nociception.

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.

Phenelzine-based probes reveal Secernin-3 is involved in thermal nociception.

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.

Reconstitution of human adrenocortical specification and steroidogenesis using induced pluripotent stem cells.

The mechanisms leading to adrenal cortex development and steroid synthesis in humans remain poorly understood due to the paucity of model systems. Herein, we recapitulate human fetal adrenal cortex specification processes through stepwise induction of human-induced pluripotent stem cells through posterior intermediate mesoderm-like and adrenocortical 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 steroid synthesis is amplified by ACTH/PKA signaling and blocked by inhibitors of Δ5 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.

Activity-Based Hydrazine Probes for Protein Profiling of Electrophilic Functionality in Therapeutic Targets.

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.

Acyl-CoA synthetase 6 regulates long-chain polyunsaturated fatty acid composition of membrane phospholipids in spermatids and supports normal spermatogenic processes in mice.

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.

Prostaglandin E2 inhibits neutrophil extracellular trap formation through production of cyclic AMP.

BACKGROUND AND PURPOSE: Upon 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 PGE2 , a bioactive substance generated in inflammatory tissues, in the formation of NETs remains unclear. EXPERIMENTAL APPROACH: The effects of PGE2 , 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 RESULTS: PGE2 inhibited PMA-induced NET formation in vitro through EP2 and EP4 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 EP2 receptor agonist. CONCLUSIONS AND IMPLICATIONS: PGE2 inhibits NET formation through the production of cAMP. These findings will contribute to the development of novel treatments for NETosis-related diseases.