A journey toward Bioorthogonal Profiling of Protein Methylation inside living cells

Abstract

Human protein methyltransferases (PMTs) play essential roles through methylating histone and nonhistone targets. It is very challenging to profile global methylation (or methylome) in the context of relevant cellular settings. Unlike other posttranslational modifications such as lysine acetylation or Ser/Thr/Tyr/His phosphorylation, methylation of lysine or arginine does not significantly alter its physical properties (e.g. charge and size) and therefore may not be probed readily by conventional biological tools such antibodies. It is also not trivial to assign unambiguously dynamic methylation events to specific PMTs given their potential redundancy. This review focuses on the decade-long progress in developing complementary chemical tools to elucidate targets of designated PMTs. One of such efforts was to develop the Bioorthogonal Profiling of Protein Methylation (BPPM) technology in vitro and inside living cells.

Functional AdoMet Isosteres Resistant to Classical AdoMet Degradation Pathways

“…13–15,27,28 While there have been great advances in methylation-dependent bioinformatics and disease-associated biomarkers, 29–31 the study of intracellular MT spatial/temporal resolution, specificity, and/or function remains a challenge. 9,14,15,25–28,32–36 Toward this end, the pioneering demonstration of AdoMet analogs, bearing alternative alkyl donor substituents, as cosubstrates for DNA 37 or natural product (NP) 38 MTs has inspired new tools and strategies to study NP, 39–42 protein, 43–51 and nucleic acid 6,52–57 methylation where the recent development of enzyme-based strategies for the synthesis of differentially alkylated AdoMet analogs has simplified access to these unique cosubstrates. 42,49,50,57–59 However, the stability of AdoMet or its corresponding differentially alkylated analogs under physiological conditions limits their utility as reagents or therapeutics by virtue of two fundamental degradative processes: intramolecular cyclization to homoserine lactone and 5′-deoxy-5′-(alkylthio)adenosine (Figure 1, pathway a) and depurination (Figure 1, pathway b).…”

AdoMet analog synthesis and utilization: current state of the art

“…‘Bump-and-hole’ technologies [78], such as those pioneered by Shokat and colleagues [79], serve as the basis for similar AdoMet adenine-modified strategies to achieve MT bioorthogonality as exemplified by the early work of Schultz and Gray [80] and a more recent example by the Zhou group [81]. Alternatively, Luo and collaborators have pursued putative bioorthogonality via targeting specific AdoMet S -alkyl modifications [17,67]. This growing precedent suggests a vibrant future for cell-based, and possibly even whole animal, applications where the fundamental key to achieving true bioorthogonality will depend on the development of AdoMet surrogate/catalyst pairings that display suitable selectivity for the targeted process/reaction over native biochemical processes/enzymes [82].…”

“…As with the nucleic acid strategies highlighted in the previous section, AdoMet analogs also enable selective installation of novel chemoselective handles to track and identify methylation events catalyzed by PMTs [12–18,57–66], the proof of concept of which was first demonstrated by Weinhold and coworkers using the wild-type PMT Dim-5 and AdoMet analog 18 (Table 1) [60]. Interestingly, while 18 is also a validated substrate of other wild-type PMTs and NAMTs [50,53], Luo and collaborators more recently reported the need for engineered PMTs to accommodate this AdoMet analog in the pursuit of putative bioorthogonal reagents to study PMT-catalyzed methylation events in vitro and living cells [14,17,18,59,63,65,67]. Isotopic tags have also been installed using corresponding PMTs and suitably-labeled AdoMet analogs (Table 1, entries 1 – 3 ) [68–73].…”

Allele-Specific Chemical Genetics: Concept, Strategies, and Applications

“…101 The central component of the approach was to engineer the active site of a PMT to create a space to accommodate and catalytically transfer the bulky sulfonium group present in a SAM analogue to substrate molecules ( Figure 8C). In a series of publications, the Luo group developed such an allele-specific catalytic system for multiple PMTs and SAM analogues.…”