Proof of concept: Pull down assay using bovine serum albumin and an immunomodulator small molecule.

In the past decade, there has been increasing interest in use of small molecules for immunomodulation. The affinity-based pull-down purification is an essential tool for target identification of small molecules and drug discovery. This study presents our recent efforts to investigate the cellular target(s) of Compound A, a small molecule with demonstrated immunomodulatory properties in human peripheral blood mononuclear cells (PBMCs). While we have previously observed the immunomodulatory activity of Compound A in PBMCs, the specific molecular targets underlying its effects remains elusive. To address this challenge, we synthesized a trifluoromethyl phenyl diazirine (TPD)-bearing trifunctional Probe 1 based on the chemical structure of Compound A, which could be used in a pull-down assay to efficiently bind to putative cellular targets via photoaffinity labelling. In this report, we utilized bovine serum albumin (BSA) as a model protein to establish a proof-of-concept in order to assess the suitability of Probe 1 for binding to an endogenous target. By the successful synthesis of Probe 1 and demonstrating the efficient binding of Probe 1 to BSA, we propose that this method can be used as a tool for further identification of potential protein targets of small molecules in living cells. Our findings provide a valuable starting point for further investigations into the molecular mechanisms underlying the immunomodulatory effects of Compound A.

A molecular toolbox for studying protein degradation in mammalian cells.

Protein degradation is a crucial regulatory process in maintaining cellular proteostasis. The selective degradation of intracellular proteins controls diverse cellular and biochemical processes in all kingdoms of life. Targeted protein degradation is implicated in controlling the levels of regulatory proteins as well as eliminating misfolded and any otherwise abnormal proteins. Deregulation of protein degradation is concomitant with the progression of various neurodegenerative disorders such as Parkinson's and Alzheimer's diseases. Thus, methods of measuring metabolic half-lives of proteins greatly influence our understanding of the diverse functions of proteins in mammalian cells including neuronal cells. Historically, protein degradation rates have been studied via exploiting methods that estimate overall protein degradation or focus on few individual proteins. Notably, with the recent technical advances and developments in proteomic and imaging techniques, it is now possible to measure degradation rates of a large repertoire of defined proteins and analyze the degradation profile in a detailed spatio-temporal manner, with the aim of determining proteome-wide protein stabilities upon different physiological conditions. Herein, we discuss some of the classical and novel methods for determining protein degradation rates highlighting the crucial role of some state of art approaches in deciphering the global impact of dynamic nature of targeted degradation of cellular proteins. This article is part of the Special Issue "Proteomics".

2-Methoxyestradiol protects against pressure overload-induced left ventricular hypertrophy.

Numerous experimental studies have supported the evidence that 2-methoxyestradiol (2 ME) is a biologically active metabolite that mediates multiple effects on the cardiovascular system, largely independent of the estrogen receptor. 2 ME is a major cytochrome P450 1B1 (CYP1B1) metabolite and has been reported to have vasoprotective and anti-inflammatory actions. However, whether 2 ME would prevent cardiac hypertrophy induced by abdominal aortic constriction (AAC) has not been investigated yet. Therefore, the overall objectives of the present study were to elucidate the potential antihypertrophic effect of 2 ME and explore the mechanism(s) involved. Our results showed that 2 ME significantly inhibited AAC-induced left ventricular hypertrophy using echocardiography. The antihypertrophic effect of 2 ME was associated with a significant inhibition of CYP1B1 and mid-chain hydroxyeicosatetraenoic acids. Based on proteomics data, the protective effect of 2 ME is linked to the induction of antioxidant and anti-inflammatory proteins in addition to the modulation of proteins involved in myocardial energy metabolism. In vitro, 2 ME has shown a direct antihypertrophic effect through mitogen-activated protein kinases- and nuclear factor-κB-dependent mechanisms. The present work shows a strong evidence that 2 ME protects against left ventricular hypertrophy. Our data suggest the potential of repurposing 2 ME as a selective CYP1B1 inhibitor for the treatment of heart failure.