Muhammad Jbara

The first PeT-based turn-on probe utilizing a chemiluminescent mechanism is presented. The probe is activated through a reaction between a quenched dioxetane-azide and a strained cycloalkyne, resulting in high signal-to-noise protein detection and outperforming a fluorescent analog.

Abstract

PeT-based fluorescent probes were demonstrated to be powerful tools for detection and imaging, owing to their significant fluorescence enhancement in response to specific targets. While numerous examples of fluorescence-based PeT have been frequently reported, there is not even a single example of a PeT probe that operates via a chemiluminescence mode. Here we report the first PeT-based turn-on probe that acts via a chemiluminescent operation mode. We designed, synthesized, and evaluated a novel chemiluminescent probe, featuring a PeT-based turn-on mechanism. The probe consists of a phenoxy-1,2-dioxetane, linked to an azide unit that acts as a PeT quencher. Upon cycloaddition of a strained cycloalkyne with the azide, a triazole-dioxetane is formed, which undergoes relatively slow chemiexcitation, resulting in a measurement window with an exceptionally high signal-to-noise ratio (over 5000-fold). The PeT-dioxetane probe could effectively detect and image two model proteins labeled with strained cycloalkyne units (Myc-DBCO and Max-DBCO) through either NHS or maleimide conjugations. Comparative analysis shows that our PeT-based chemiluminescent probe significantly outperforms a commercially available fluorescent analog. We anticipate that the insights gained from this study will facilitate the development of additional chemiluminescent probes utilizing various PeT-quenching pathways.

A library of Max transcription factor variants with distinct modifications was prepared by a practical strategy to probe the impact of Ser phosphorylation. The phosphorylation pattern was found to play a crucial role in DNA-binding activity but did not significant affect sequence specificity. The insight gained on the regulatory role of Max phosphorylation sheds light on how post-translational modifications influence transcription factor activity.

Abstract

The chemical synthesis of site-specifically modified transcription factors (TFs) is a powerful method to investigate how post-translational modifications (PTMs) influence TF-DNA interactions and impact gene expression. Among these TFs, Max plays a pivotal role in controlling the expression of 15 % of the genome. The activity of Max is regulated by PTMs; Ser-phosphorylation at the N-terminus is considered one of the key regulatory mechanisms. In this study, we developed a practical synthetic strategy to prepare homogeneous full-length Max for the first time, to explore the impact of Max phosphorylation. We prepared a focused library of eight Max variants, with distinct modification patterns, including mono-phosphorylated, and doubly phosphorylated analogues at Ser2/Ser11 as well as fluorescently labeled variants through native chemical ligation. Through comprehensive DNA binding analyses, we discovered that the phosphorylation position plays a crucial role in the DNA-binding activity of Max. Furthermore, in vitro high-throughput analysis using DNA microarrays revealed that the N-terminus phosphorylation pattern does not interfere with the DNA sequence specificity of Max. Our work provides insights into the regulatory role of Max′s phosphorylation on the DNA interactions and sequence specificity, shedding light on how PTMs influence TF function.

Protein chemistry has evolved remarkably in the past decade, which has enabled the effective production of new molecules with novel physicochemical properties for biomedical research, and therapeutic applications. This minireview summarizes recent developments in chemical protein synthesis to produce bioactive proteins, with emphasis on novel analogs with promising in vitro and in vivo activity.

Abstract

Nature has developed a plethora of protein machinery to operate and maintain nearly every task of cellular life. These processes are tightly regulated via post-expression modifications—transformations that modulate intracellular protein synthesis, folding, and activation. Methods to prepare homogeneously and precisely modified proteins are essential to probe their function and design new bioactive modalities. Synthetic chemistry has contributed remarkably to protein science by allowing the preparation of novel biomacromolecules that are often challenging or impractical to prepare via common biological means. The ability to chemically build and precisely modify proteins has enabled the production of new molecules with novel physicochemical properties and programmed activity for biomedical research, diagnostic, and therapeutic applications. This minireview summarizes recent developments in chemical protein synthesis to produce bioactive proteins, with emphasis on novel analogs with promising in vitro and in vivo activity.

Nature, Published online: 26 October 2020; doi:10.1038/d41586-020-02991-1

Oxidation of methionine residues in proteins is a biologically important post‐translational modification that is difficult to study. We show that activation of sulfoxides with TMSCl generates α‐chloro intermediates, which then react with specific thiols to generate dithioacetals. This modified Pummerer reaction can be used to study methionine sulfoxides in proteins.

Abstract

The reversible oxidation of methionine residues in proteins has emerged as a biologically important post‐translational modification. However, detection and quantitation of methionine sulfoxide in proteins is difficult. Our aim is to develop a method for specifically derivatizing methionine sulfoxide residues. We report a Pummerer rearrangement of methionine sulfoxide treated sequentially with trimethylsilyl chloride and then 2‐mercaptoimidazole or pyridine‐2‐thiol to produce a dithioacetal product. This derivative is stable to standard mass spectrometry conditions, and its formation identified oxidized methionine residues. The scope and requirements of dithioacetal formation are reported for methionine sulfoxide and model substrates. The reaction intermediates have been investigated by computational techniques and by ¹³C NMR spectroscopy. These provide evidence for an α‐chlorinated intermediate. The derivatization allows for detection and quantitation of methionine sulfoxide in proteins by mass spectrometry and potentially by immunochemical methods.

Identification of protein S‐glutathionylation is important for understanding redox‐mediated biological processes that are regulated by reactive oxygen species. Isotopically labeled clickable glutathione was developed and applied for relative quantification of glutathionylated peptides upon addition of hydrogen peroxide to a cardiomyocyte cell line.

Abstract

Protein S‐glutathionylation is one of the important cysteine oxidation events that regulate various redox‐mediated biological processes. Despite several existing methods, there are few proteomic approaches to identify and quantify specific cysteine residues susceptible to S‐glutathionylation. We previously developed a clickable glutathione approach that labels intracellular glutathione with azido‐Ala by using a mutant form of glutathione synthetase. In this study, we developed a quantification strategy with clickable glutathione by using isotopically labeled heavy and light derivatives of azido‐Ala, which provides the relative quantification of glutathionylated peptides in mass spectrometry‐based proteomic analysis. We applied isotopically labeled clickable glutathione to HL‐1 cardiomyocytes, quantifying relative levels of 1398 glutathionylated peptides upon addition of hydrogen peroxide. Importantly, we highlight elevated levels of glutathionylation on sarcomere‐associated muscle proteins while validating glutathionylation of two structural proteins, α‐actinin and desmin. Our report provides a chemical proteomic strategy to quantify specific glutathionylated cysteines.