Desuccinylation-Triggered Peptide Self-Assembly: Live Cell Imaging of SIRT5 Activity and Mitochondrial Activity Modulation

Abstract

Mimicking nature's ability to orchestrate molecular self-assembly in living cells is important yet challenging. Molecular self-assembly has found wide applications in cellular activity control, drug delivery, biomarker imaging, etc. Nonetheless, examples of suborganelle-confined supramolecular self-assembly are quite rare and research in this area remains challenging. Herein, we have presented a new strategy to program supramolecular selfassembly specifically in mitochondria by leveraging on a unique enzyme SIRT5. SIRT5 is a mitochondria-localized enzyme belonging to a family of NAD + -dependent histone deacetylases. Accumulating studies suggest that SIRT5 is involved in regulating diverse biological processes, such as reactive oxygen defense, fatty acid metabolism, and apoptosis. In this study, we designed a novel class of succinylated peptide precursors that can be transformed into self-assembling building blocks through SIRT5 catalysis, leading to the formation of supramolecular nanofibers in vitro and in living cells. The increased hydrophobicity arising from self-assembly remarkably enhanced the fluorescence of nitrobenzoxadiazole (NBD) in the nanofibers. With this approach, we have enabled activity-based imaging of SIRT5 in living cells for the first time. Moreover, SIRT5-mediated peptide self-assembly was found to depolarize mitochondria membrane potential and promote ROS formation. Coincubation of the peptide with three different chemotherapeutic agents significantly boosted the anticancer activities of these drugs. Our work has thus illustrated a new way of mitochondria-confined peptide self-assembly for SIRT5 imaging and potential anticancer treatment.

Biological functions of supramolecular assemblies of small molecules in the cellular environment

“… 57 This is the first report to employ enzymatic noncovalent synthesis 24 for targeting mitochondria, complementing the “TPP” approach. 56 This approach should be applicable to the use of other enzymes and to inhibit other cancer cells, which also stimulates efforts to design more precursors that allow enzymatic reactions to generate SASMs at mitochondria of cancer cells, as shown by the recent work of Sun et al 59 In addition, the process of forming SASMs catalyzed by ENTK also finds applications for delivering genes selectively into mitochondria of cancer cells. 60 …”

Supramolecular peptide nano-assemblies for cancer diagnosis and therapy: from molecular design to material synthesis and function-specific applications

“…Recently, Yang and co-workers reported the desuccinylation-triggered formation of peptide nanofibers for liver cell imaging[ 83 ] As shown in Fig. 8 a, a fluorescence module is conjugated onto self-assembling peptide sequence for the design of a functional peptide molecules, which could self-assemble into nanofibers at pH 7.4 under the catalysis of SIRT5, mitochondria-localized enzyme.…”

“…Reprinted with permission from Ref. [ 83 ], Copyright 2020, American Chemical Society. (d) PNF-GQD nano-assemblies for imaging.…”

Triple Enzyme‐Regulated Molecular Hydrogels for Carrier‐Free Delivery of Lonidamine

“…To verify the expression levels of ALP and CES, we screened a series of mammalian cells (cancerous cells A375 and HeLa, normal cells HaCaT and NIH3T3) according to previously established protocols. [14] First, 4-nitro-2,1,3-benzoxadiazole (NBD)-capped substrate of phosphatase (NBD-1p) was synthesized (Figure S10, Supporting Information), and the expression of ALP was visualized by intracellular fluorescence of NBD, [15] for dephosphorylation of NBD-1p triggered the self-assembly to give bright fluorescence signals of NBD. Examinations with confocal laser microscopy (CLMS) revealed that relative fluorescence intensities of NBD-1p-treated cells were 2.3, 2.1, 0.8, and 1.0 for A375, HeLa, HaCaT, and NIH3T3 cells separately (Figure S11, Supporting Information), suggesting cancerous cells A375 and HeLa exhibited higher expression levels of ALP.…”