Enzymatic Noncovalent Synthesis for Targeting Subcellular Organelles

Abstract

Enzymatic noncovalent synthesis (ENS) exploits enzymatic reactions to produce spatially organized higher-order supramolecular assemblies that modulate cellular processes. While ENS is a general mechanism to create higher-order assemblies of proteins for diverse cellular functions, the exploration of ENS of other bioactive molecules, such as peptides or small organic molecules, is rather limited. Since ENS generates non-diffusive supramolecular assemblies locally, it provides a unique approach to targeting subcellular organelles. In this review, we highlight the recent progress of the application of ENS of peptide assemblies for targeting subcellular organelles. After a brief introduction of the concept of ENS, we introduce the case of generating artificial filaments by ENS in cell cytosol, then discuss the use of ENS for targeting endoplasmic reticulum, mitochondria, Golgi apparatus, and lysosomes, and finally we describe the targeting of nucleus by ENS. We hope to illustrate the promise of ENS, as a localized molecular process in an open system, for understanding diseases, controlling cell behaviors, and developing new therapeutics.

Graphical Abstract

This review highlights the recent progress of the application of enzymatic noncovalent synthesis (ENS) of peptide assemblies for targeting subcellular organelles, including the generation of artificial filaments in cytosol, targeting endoplasmic reticulum, mitochondria, Golgi apparatus, lysosomes, and nucleus. We hope to illustrate the promise of ENS, as a localized molecular process in an open system, for understanding diseases, controlling cell behaviors, and developing new therapeutics.

1. Introduction

Enzymatic noncovalent synthesis (ENS) refers to a molecular process that integrates enzymatic reactions and self-assembly for spatiotemporal organization of higher-order molecular assemblies.^[1] ENS of protein assemblies controls cell physiology,^[2] demonstrating that noncovalent assembly is an integrated behavior of cells.^[1] These facts illustrate the essence of ENS—that is, enzymatic reactions control the locations of supramolecular assemblies that regulate cellular functions. While ENS of proteins is a general mechanism to form spatially organized higher-order protein assemblies for cellular functions, the exploration of cellular ENS of the assemblies of synthetic molecules, however, remains largely unexplored. Peptide assemblies^[3] have been investigated in a few biomedical contexts,^[3–4] such as scaffolds for tissue engineering^[4a] or as drug carriers.^[4d] Recent studies on peptide assemblies demonstrate that ENS is particularly effective to generate non-diffusive peptide assemblies for modulating cellular functions. Particularly, ENS of peptide assemblies is able to target certain subcellular organelles. These unexpected and exciting findings demonstrate the promise of ENS, but the applications of ENS of peptide assemblies for targeting subcellular remain to be further explored.

In this review, we highlight the recent progress of the use of ENS of peptide assemblies for targeting subcellular organelles. We apologize for not being able to include many excellent works^[5] due to the limited space. We first give a brief description of the concept of ENS, then give the update on generating artificial peptide filaments by ENS in cell cytosol. Following that, we discuss the use of ENS for targeting endoplasmic reticulum, mitochondria, Golgi apparatus, and lysosomes, and finally we describe the targeting of nucleus by ENS. We hope to illustrate the prospect of ENS, as a localized molecular process, for understanding diseases, controlling cell behaviors, and developing new therapeutics.

2. The concept of ENS

As shown in Figure 1, ENS, as an out-of-equilibrium process, converts many molecules (either individually or in ensembles) to the assemblies of new molecules by enzymatic reaction and self-assembly. Because the resulting assemblies are non-diffusive, ENS acts as a localized molecular process to control noncovalent interactions and to define emergent properties of molecular ensembles, especially in a complicated open system like cells, which is dynamic and responsive to the external environment in addition to substance/energy exchange in other biological open systems. The functions of the assemblies from ENS usually depend on the history of molecular transformations. For example, peptide assemblies can inhibit cells when being made by ENS, but the assemblies of the same peptide can be innocuous to cells when being formed by self-assembly only.^[6] Thus, ENS utilizes reactions to confer new functions on peptide assemblies. This feature differs ENS from conventional self-assembly,^[7] which does not generate new molecules. Compared to conventional noncovalent synthesis, which is carried out in non-aqueous solvents or non-physiological conditions, such as in organic solvents or at high temperatures, ENS of peptide assemblies is well suited to take place in the cellular environment.

Figure 1.

The relationship among self-assembly, ECS, and ENS and the corresponding targets and products. Adapted with permission from Ref^[1]. Copyright 2020 American Chemical Society.

Because peptide assemblies are non-diffusive or have low diffusivity, ENS of peptide assemblies provides a unique way to control cellular functions based on the localized reactions catalyzed by enzymes. For example, with proper substrates of certain enzymes, ENS forms peptide assemblies either inside cells^[8] or on the cell surface^[6], even in the case of single cells.^{[6, 9]} Thus, cellular ENS provides spatiotemporal control of peptide assemblies for targeting subcellular organelles. Figure 2 shows the molecular structures of the peptides for ENS with various subcellular targets to be discussed in this review. The spatiotemporal control provided by ENS endows the resulting non-diffusive peptide assemblies with new functions and provides new strategies to address some of the most vexing biomedical challenges, including mitochondrial delivery^[10] and generation of artificial intracellular filaments^[11].

Figure 2.

Molecular design of peptides for ENS discussed in this review.

3. Intracellular artificial filaments formed by ENS

Although the concept of forming intracellular peptide assemblies by ENS has been demonstrated one and half decade ago,^[12] the unambiguous demonstration of intracellular artificial filaments is rather recent. As shown in Figure 3, a D-phosphotetrapeptide (1), bearing trimethylated L-lysine at the C-terminal, self-assembles to form nanoparticles. These nanoparticles, apparently forming liquid condensates inside cells, become bundles of the filaments of peptides (2) upon dephosphorylation catalyzed by alkaline phosphatase (ALP). The dephosphorylated peptide (2) self-assembles into two distinct types of cross-β structures, as revealed by cryo-EM helical reconstructions using IHRSR.^[13] Electron tomography confirms that twisted bundles of the peptide filaments form inside cells. Structural analogs of 1 indicate that heterochiral peptide backbone and the trimethylation of the lysine are critical for forming intracellular bundles of the filaments, although all the analogs form nanofibers upon dephosphorylation in cell-free conditions. This observation highlights the inherent differences between cell-free and in cellulo conditions, that is, cell is a dynamic open system with ubiquitous exchange of biological molecules and energy with the surroundings to tightly regulate the cellular environment. This work, besides providing insights into generating artificial intracellular filaments, illustrates that enzymatic morphological transition (nanoparticle-to-nanofiber) is a feasible approach to creating higher-order structures of peptide assemblies inside cells for modulating cellular functions.

Figure 3.

A) TEM images of nanostructures formed by 1 before and after the addition of 2 U/mL ALP to 200 μM of 1 for 24 h. B) A cryo-EM image of type 1 (yellow arrow) and type 2 (red arrow) filaments of 2 and 3D reconstruction of the filaments from cryo-EM images. C) Schematic illustration of the intracellular formation of artificial filaments. Adapted from Ref^[11].

4. Targeting Mitochondria

Mitochondria are complex organelles that play a central role in key cellular processes, particularly in acting as the hub for bioenergetic, biosynthetic, and signaling events.^[14] Delivering genes to mitochondria holds great potential in biomedicine because mitochondria are one of the most important drug targets for treating a wide range of diseases, including cancer, cardiovascular, and neurological disorders. The current strategies for targeting mitochondria, however, lack efficiency or cell specificity, thus may result in unwanted side effects. Therefore, it is necessary to explore new approaches to delivering genes into mitochondria efficiently in a cell type-specific manner. Mitochondrial ENS is able to deliver cargos (including genes) to mitochondria efficiently in a cell type-specific manner (Figure 4),^[15] that is, the peptide (3) self-assembles to form micelles. When enterokinase (ENTK) catalytically cleaves the peptide, the micelles turn into nanofibers of 4. When this process occurs on mitochondria of certain cancer cells, ENS of peptides enables cancer-selective mitochondrial genetic engineering.^[10] Specifically, 3, as the substrate of mitochondrial ENS, is able to transfect gene vectors encoding CRISPR/Cas9^[16] into the mitochondria of cancer cells to knockout MT-CO1 gene. This mitochondria ENS also facilitates the gene expression of FUNDC1 and GFP-tagged p53 proteins in the mitochondria of cancer cells, which induce mitophagy^[17] and apoptosis^[14a], respectively. Moreover, this ENS process results in the exclusive expression of non-mitochondrial proteins (e.g., GFP, RFP-LAMP1, or GFP-PTS) in cancer cell mitochondria. This work, by illustrating mitochondrial ENS for assisting genetic engineering of cancer cell mitochondria, underscores the promises of ENS of peptide assemblies for targeting mitochondria. Recently, Sun et al. showed that SITR5 is able to catalyze mitochondrial ENS of succinylated peptides for inhibiting cancer cells.^[18] In another study, Ryu et al. reported that pyrene-conjugated peptides form intramitochondrial assemblies for inhibiting cancer cells in vitro and in vivo. They found that both D- and L-isomeric forms exhibit similar activities, which implies that the inhibitory activities originate from rather the assemblies than individual molecules.^[5d]

Figure 4.

Schematic illustration of mitochondrial ENS. A) Illustration of ENTK-catalyzed self-assembly of 4.B) Illustration of the mitochondrial ENS of 3 facilitating mitochondrial genetic engineering. Adapted from Ref^[10].

5. Targeting Endoplasmic Reticulum

Endoplasmic reticulum (ER) is the largest cellular organelle and plays critical roles in many processes, including biosynthesis, sensing, and signaling, especially in eukaryotic cells. It is well established that disrupting normal protein-folding capacity of ER leads to ER stress that can eventually result in cell death. It is, however, difficult to only cause ER stress in cancer cells, considering that cancer cells may evolve cellular processes to decrease ER stress. ENS to generate peptide assemblies at ER is a new strategy to target ER of cancer cells.^[19] For example, a heterochiral phosphotetrapeptide (5) disrupts cell membranes and targets ER to result in cancer cell death (Figure 5). The self-assembly of 5 forms twisted rods, which become crescent-shaped aggregates of 6 after dephosphorylation catalyzed by ALP. The aggregates of 6 are to interact with lipid membranes to enter cancer cells. The assemblies of 1, being taken up by the cancer cells, eventually accumulate at the ER and induce ER stress of cancer cells, which leads to apoptosis. Structural exploration of the analogues of 5 suggests that L-homoarginine at the C-terminal of the D-tripeptide likely is responsible for the crescent-shaped morphology of the assemblies of 6. Late examples of ER targeting by peptide assemblies^{[19b, 19d]} are able to selectively induce the death of cancer cells without the need of heterochiral peptides. These results imply that ENS of peptide assemblies provides diverse ways to target ER.

Figure 5.

A) Schematic illustration of peptide assemblies generated via ENS disrupting the cell membrane and targeting ER. B) HRTEM images of nanostructures before and after the addition of 1 U/mL of ALP to 0.5 wt% of 5. Adapted with permission from Ref^[19a]. Copyright 2018 American Chemical Society.

6. Targeting Golgi Apparatus

Golgi apparatus (GA) is the “traffic center” of cells^[20] and acts as a key signaling hub of cells.^[21] Although it is attractive to target GA to induce the death of cancer cells, it is challenging to selectively target GA of cancer cells. A recent study shows that ENS may provide a way to target GA of cancer cells.^[22] When the oxygen atom of the phosphoester bond in a phosphopeptide is replaced by a sulfur atom to make 7, the thiophosphopeptide (7) not only undergoes fast enzymatic self-assembly catalysed by ALP, but also forms the thiopeptide (8), which is able to target GA of cancer cells that overexpress ALP. For example, the incubation of 7 with HeLa cells results in almost instant accumulates of 8 at GA of the HeLa cells (Figure 6), even when the concentration of 7 is as low as 500 nM. Similar fast enzymatic accumulation of 8 also takes place in the GA of several other cells (e.g., Saos-2, SJSA-1, OVSAHO, HCC1937, and HEK293) that overexpress ALP. Further mechanistic studies reveal several underappreciated features of the ENS of thiophosphopeptides, such as the thiophosphopeptides entering cells via multiple pathways (e.g., caveolin-mediated endocytosis and micropinocytosis), oxidative dimerization of 8 for the GA targeting, and GA accumulation correlating well with the ALP expression. This work is the first example that combines ENS with redox responsive molecules for targeting subcellular organelles. Moreover, the same study reported that the thiophosphopeptides potently and selectively inhibit cancer cells (e.g., HeLa) with a low IC₅₀ (about 3 μM), likely by activating multiple cell death pathways or by enabling a new cell death mechanism. Further exploration along this concept will be fruitful.

Figure 6.

A) Schematic illustration of 7 targeting Golgi apparatus instantly by ENS and subsequent formation of disulfide bonds. B) Confocal laser scanning microcopy images of Golgi-RFP-stained HeLa cells after treatment with 7 for 8 min. Adapted with permission from Ref^[22]. Copyright 2021 Wiley.

7. Targeting Lysosome

A common subcellular organelle for processing molecular assemblies is lysosome. Several elegant works reported the use of ENS for targeting lysosomes based on lysosomal enzymes. For example, Yang et al. reported a tandem enzymatic self-assembly approach to target lysosomes. Taking the advantage of an A549 lung cancer cell line overexpressing extracellular ALP and intracellular reductase, the authors designed a substrate 9 that contains a tyrosine phosphate and an azobenzene to respond to ALP and the reductase, respectively.^[5b] This design results in tandem molecular self-assembly of nanofibers, which first targets lysosomes. After lysosomal escape, the nanofibers further disrupt mitochondrial membrane to generate ROS, thus resulting in severe endoplasmic reticulum (ER) stress (Figure 7A). With the dosage of 5 mg/kg, the substrate selectively inhibits the lung cancer cells in vitro and A549 xenograft tumors in vivo. This work illustrates that ENS catalyzed by multiple enzymes may lead to promising nanomedicine for the diagnosis and treatment of lung cancer. In a recent study, Wang et al. report an elegant way to employ cathepsin B (CTSB), which is often overexpressed in the lysosomes of cancer cells, for ENS and inhibiting tumor growth.^[23] The authors hypothesized that CTSB could initiate ENS after cellular uptake of the precursor, thus selectively generating cytotoxic assemblies in cancer cells (Figure 7B). They developed a CTSB substrate (12), a peptide precursor that contains a CTSB cleavage site and C-terminal glycosylation. Using a series of techniques, including confocal laser scanning microscopy, biological electron microscopy imaging, western blot, flow cytometry, and structure activity relationship (SAR), the authors established that 12 undergoes ENS to form higher-order structures inside the lysosomes. This process results in lysosomal membrane permeabilization (LMP), which induces glioblastoma cancer cell death selectively through necroptosis (Figure 7B). The use of C-terminal glycosylation in this work is particularly exciting as it illustrates the broad molecular space for ENS. In another work, Ding et al. reported turning immunologically cold tumors hot by LMP-induced ENS.^[24]

Figure 7.

A) Schematic illustration of tandem molecular self-assembly of 9 targeting lysosomes, disrupting mitochondrial membrane and inducing endoplasmic reticulum stress. Adapted from Ref^[5b]. B) Schematic illustration of the formation of higher-order assemblies of 12 inside lysosomes for selective cell inhibition. Adapted with permission from Ref^[23]. Copyright 2021 Wiley.

8. Targeting Nucleus

Nucleus, which stores the genes of cells and acts as the control center of the cell, probably is the most important subcellular organelle. But it is challenging to target nucleus by ENS, unless the building blocks of the ENS substrates are intercalators for DNA, as first reported by Yang et al.^[25] An unexpected report of targeting nucleus by ENS is the use of ENS for selectively killing human induced pluripotent stem cells (iPSCs).^[26] Despite of iPSCs’ promise in revolutionizing personalized medicine,^[27] the tumorigenicity^[28] of iPSCs remains a major safety concern. Because iPSCs overexpress (or upregulate) ALP,^[29] it is possible to use ALP to catalyze ENS for selectively killing iPSCs.^[30] To minimize the potential side effects associated with D-phosphopeptides, Liu et al. developed an L-phosphopentapeptide (13), which forms α-helix and aggregated strands of 14 upon the dephosphorylation catalyzed by ALP. The surprise is that the peptide assemblies of 14 form inside nucleus (Figure 8) for selectively killing iPSCs. The phosphopentapeptide made of four L-leucine residues and a C-terminal L-phosphotyrosine forms micelles or nanoparticles. The micelles turn into nanoribbons after rapid enzymatic dephosphorylation. The high expression of ALP and the structure of 13 both contribute to the nucleus targeting, but the detailed mechanism for forming intranuclear nanoribbons of 14 in iPSCs remains to be elucidated. Further elucidating this phenomenon may lead to more effective ENS substrates for targeting nucleus.

Figure 8.

A) Schematic illustration of intranuclear ENS of 14 from 13. B) Confocal laser scanning microscopy images of iPSCs treated with 400 μM of 13 for 2 h to reveal the intranuclear assemblies. Adapted with permission from Ref^[26]. Copyright 2021 American Chemical Society.

8. Conclusion and Outlook

ENS of peptide assemblies allows new types of questions (i.e., the functions of localized enzymatic reactions and self-assembly) to be asked, which could not be anticipated in the absence of ENS. This prospect is exciting because it likely would lead to new discoveries. In fact, none of the examples of targeting subcellular organelles discussed above results from a priori designs. This fact highlights the promises of ENS. Moreover, ENS generates supramolecular assemblies of peptides inside cells. Considering that cells are a type of open system that dissipates energy, ENS provides a feasible and unique approach to explore self-assembly or self-organization in open systems. Since the early demonstration of the anticancer activities of peptide assemblies in cell assays^[12a] and in a murine model^[31], there has been an increasing number of reports about the molecular building blocks for ENS and the identification of new functions. Future studies would pay more attention to the mechanisms of the actions of ENS of peptide assemblies inside cells, as well as the atomistic structures of the peptide assemblies^[32] and in vivo (i.e., animal models) test of ENS^[33]. Although the pharmacodynamic and pharmacokinetic properties of the molecules can only be assessed in animal models, the knowledge of ENS in cell assays provides a necessary understanding of molecule-cell interactions, which is the foundation of animal model studies. To overcome the challenges in elucidating the mechanisms, a multidisciplinary collaboration among chemists, biologists, engineers, and medical scientists would be necessary. Nature has evolved ENS in cells. Now it is time for molecular scientists to learn the essence of ENS for advancing molecular science, which would help understand nature and address societal problems.

Biographies

Author biographies

Qiuxin Zhang obtained her BEng degree in Nanomaterials and Nanotechnology from the College of Nano Science and Technology, Soochow University and BS degree in Materials and Nanosciences from University of Waterloo both in 2019. She is currently a third-year graduate student supervised by Prof. Bing Xu in the Department of Chemistry at Brandeis University. Her research interest focuses on peptide assemblies and their promise in cancer therapeutics.

Weiyi Tan obtained his BS degree from the College of Chemistry, Chemical Engineering and Material Science, Soochow University, in 2018. He is currently in his fourth year as a graduate student in chemistry supervised by Professor Bing Xu at Brandeis University. His current research interest lies in designing self-assembling materials for biological applications.

Prof. Bing Xu, after receiving his BS and MS from Nanjing University in 1987 and 1990, respectively, obtained his PhD, under the supervision of Professor Timothy Swager, in 1996 from the University of Pennsylvania. Before starting his independent research at the Hong Kong University of Science and Technology from 2000, he was an NIH postdoctoral fellow in the Whitesides lab at Harvard University. He was tenured as an associate professor in Jan 2006 and became a full professor in Jul 2008 at HKUST. He is currently a professor at the Department of Chemistry, Brandeis University. His current research focuses on the applications of enzymatic noncovalent synthesis in materials, biology, and medicine.