Instructed Assembly of Peptides for Intracellular Enzyme Sequestration

Abstract

Liquid-like droplets of biomacromolecules are emerging as a fundamental mechanism of cellular signaling, but designing synthetic mimics to form such membraneless organelles remains unexplored. Here we report the use of supramolecular assemblies of small peptides, as a mimic of biomacromolecular condensates, for intracellular sequestration of enzymes on endoplasmic reticulum (ER). Specifically, integrating a short peptide with naproxen (a nonsteroidal anti-inflammatory drug (NSAID) and a ligand of cyclooxygenase-2 (COX-2)) generates an enzymatic substrate that acts as a precursor for instructed assembly. Slowly dephosphorylating the precursors by phosphatases forms the corresponding hydrogelators in a cellular environment, which results in the supramolecular assemblies on ER. Consisting of the precursor and the hydrogelator molecules, the assemblies enable the sequestration of COX-2 and protein-tyrosine phosphatase 1B (PTP1B) on ER. Further structure-activity investigation reveals that the colocalization of COX-2 and PTP1B relies on the NSAID motif, the phosphotyrosine, and the enzymatic dephosphorylation of the precursor. This work, for the first time, illustrates the use of supramolecular processes for associating enzymes in cells and may provide insights for understanding intracellular liquid condensates and a new strategy for modulating protein-protein interactions.

Figure 1.

(A) Sol-gel transition of Npx-1P (400 µM) upon adding ALP (1 U/mL). (B) CMCs of Npx-1P and Npx-1. (C) Dephosphorylation of Npx-1P (12.5 µM) after incubating with 0.1 U/mL ALP at different time. (D) TEM images of nanostructures formed before and after adding ALP (0.1 U/mL) to the solution of Npx-1P (12.5 µM)(scale bar = 100 nm). All the solutions were prepared in pH 7.4 PBS buffer.

Figure 2.

Pull down of COX-2 and PTP1B with enzymatic assemblies formed by Npx-1P in PBS. The image shows Coomassie staining of pellet (P) and supernatant (S) fractions.

Figure 3.

(A) Dephosphorylation of Npx-1P (12.5 µM) after incubating with Saos-2 cells at different time. (B) CLSM images of Saos-2 cells treated with Npx-1P (12.5 µM) for 1 h and then stained with antibodies of PTP1B (red) and COX-2 (green). (C) The enlarged region of co-localization in (B) (scale bar = 2 μm). (D) Orthogonal Z-stack of co-localization of PTP1B and COX2.

Figure 4.

CLSM images of Saos-2 cells stained with antibodies of PTP1B (red) and COX-1/COX-2 (green) after treating with Npx-1P, Nap-1P and Npx-2P for 1 h (scale bar = 10 μm).

Scheme 1.

Illustration of instructed-assembly for intracellular sequestration of PTP1B and COX-2.