Uno, H. | Search bioRxiv

bioRxiv2024

Profiling proteins involved in peroxynitrite homeostasis using ROS/RNS conditional proteomics

Peroxynitrite (ONOO-), the product of the diffusion-controlled reaction of superoxide (O2*-) with nitric oxide (NO*), plays a crucial role in oxidative and nitrative stress and modulates key physiological processes such as redox signaling. While biological ONOO- is conventionally analyzed using 3-nitrotyrosine antibodies and fluorescent sensors, such probes lack specificity and sensitivity, making high-throughput and comprehensive profiling of ONOO--associated proteins challenging. In this study, we used a conditional proteomics approach to investigate ONOO- homeostasis by identifying its protein neighbors in cells. We developed Peroxynitrite-responsive protein Labeling reagents (Porp-L) and, for the first time, discovered 2,6-dichlorophenol as an ideal moiety that can be selectively and rapidly activated by ONOO- for labeling of proximal proteins. The reaction of Porp-L with ONOO- generated several short-lived reactive intermediates that can modify Tyr, His, and Lys residues on the protein surface. We have demonstrated the Porp-L-based conditional proteomics in immune-stimulated macrophages, which indeed identified proteins known to be involved in the generation and modification of ONOO- and revealed the endoplasmic reticulum (ER) as a ONOO- hot spot. Moreover, we discovered a previously unknown role for Ero1a, an ER-resident protein, in the formation of ONOO-. Overall, Porp-L represent a promising research tool for advancing our understanding of the biological roles of ONOO-.

biochemistry↗

bioRxiv2023

The androgen receptor in mesenchymal progenitors regulates skeletal muscle mass via Igf1 expression in male mice.

Androgens exert their effects primarily by binding to the androgen receptor (AR), a ligand-dependent nuclear receptor. While androgens have anabolic effects on skeletal muscle, previous studies reported that AR functions in myofibers to regulate skeletal muscle quality, rather than skeletal muscle mass. Therefore, the anabolic effects of androgens are exerted via extra-myofiber cells or tissues. In this context, the cellular and molecular mechanisms of AR in mesenchymal progenitors, which play a crucial role in maintaining skeletal muscle homeostasis, remain largely unknown. In this study, we demonstrated expression of AR in mesenchymal progenitors and found that targeted AR ablation in mesenchymal progenitors reduced limb muscle mass in mature adult, but not young or aged, male mice, although fatty infiltration of muscle was not affected. The absence of AR in mesenchymal progenitors led to remarkable perineal muscle hypotrophy, regardless of age, due to abnormal regulation of transcripts associated with cell death and extracellular matrix organization. Additionally, we revealed that AR in mesenchymal progenitors regulates the expression of insulin-like growth factor 1 (Igf1), and that IGF1 administration prevents perineal muscles atrophy in a paracrine manner. These findings indicate that the anabolic effects of androgens regulate skeletal muscle mass via, at least in part, AR signaling in mesenchymal progenitors. Significance statementAndrogens are essential not only for the development of male sexual characteristics but also for a range of physiological functions, including the regulation of skeletal muscle growth and function. Understanding the functionality of the androgen receptor (AR) is essential for comprehending the mechanisms through which androgens exert their effects on skeletal muscles, as these effects are mediated through AR binding. We demonstrate that AR is expressed in mesenchymal progenitors, which play a vital role in muscle homeostasis, and regulates the expression of insulin-like growth factor 1 (Igf1)--a key player in skeletal muscle growth--to control muscle mass. Our study provides significant insights into potential therapeutic strategies for addressing muscle atrophy conditions like sarcopenia.

molecular biology↗