Manipulating Cell Fates with Protein Conjugates

Abstract

The homeostasis of cellular activities is essential for the normal functioning of living organisms. Hence, the ability to regulate the fates of cells is of great significance for both fundamental chemical biology studies and therapeutic development. Despite the notable success of small-molecule drugs that normally act on cellular protein functions, current clinical challenges have highlighted the use of macromolecules to tune cell function for improved therapeutic outcomes. As a class of hybrid biomacromolecules gaining rapidly increasing attention, protein conjugates have exhibited great potential as versatile tools to manipulate cell function for therapeutic applications, including cancer treatment, tissue engineering, and regenerative medicine. Therefore, recent progress in the design and assembly of protein conjugates used to regulate cell function is discussed in this review. The protein conjugates covered here are classified into three different categories based on their mechanisms of action and relevant applications: (1) regulation of intercellular interactions; (2) intervention in intracellular biological pathways; (3) termination of cell proliferation. Within each genre, a variety of protein conjugate scaffolds are discussed, which contain a diverse array of grafted molecules, such as lipids, oligonucleotides, synthetic polymers, and small molecules, with an emphasis on their conjugation methodologies and potential biomedical applications. While the current generation of protein conjugates is focused largely on delivery, the next generation is expected to address issues of site-specific conjugation, in vivo stability, controllability, target selectivity, and biocompatibility.

Figure 1.. Manipulation of cell fates by protein conjugates.

Various types of protein conjugates have been developed as tools to manipulate cell fates by (A) regulation of intercellular interactions, (B) intervention in intracellular biological pathways, and (C) termination of cell proliferation. For each category, proteins were chemically conjugated with different functional molecules, including lipids, oligonucleotides, synthetic polymers, and other small molecules, for a wide range of biomedical applications.

Figure 2.. Different protein conjugates generated to regulate intercellular interactions.

(A) Protein-lipid conjugates have been used to modify cell surface and mediate cell-cell interactions. (B) Biotinylated proteins can induce cell-cell interactions through biotin-streptavidin bridging. (C) Protein-polysaccharide conjugates have been utilized to label the cell surface and mediate cell-cell interactions. (D) Synthetic polymers were modified with functional proteins to form hydrogels for the targeted delivery of cells to specific tissues and regulate their interactions. (E) Protein-oligonucleotides conjugates have been developed to program cell-cell interactions via oligonucleotides hybridization.

Figure 3.. Different protein conjugates generated to intervene in intracellular biological pathways.

(A) Oligonucleotides covalently conjugated to proteins or non-covalently loaded onto positively charged protein-polymer conjugates for intracellular delivery to interrupt gene expression. (B) Protein-oligonucleotides synthesized to trigger cellular signaling by receptor binding and dimerization (or oligomerization). (C) Antibodies conjugated with small-molecule PROTACs or ligands for lysosome-targeting receptors to specifically degrade signaling proteins. (D) Functional proteins modified with synthetic polymers or conjugated to polymer-based hydrogels to activate receptor-mediated signaling of target cells or tissues.

Figure 4.. Different protein conjugates synthesized to terminate cell proliferation.

(A) Cytotoxic drugs have been conjugated to the antigen-specific proteins for targeted drug delivery. (B) Cytotoxic drugs can intercalate into oligonucleotides conjugated to targeting proteins for drug delivery. (C) Polymers can be coupled to proteins to form amphipathic conjugates that self-assemble into nanoparticles for the delivery of toxic drugs.

Figure 5.. Different conjugation approaches to generate protein conjugates for the regulation of cell functions.

Most non-specific conjugations can be conducted by (A) reactions between primary amines and NHS esters or (B) reduced thiol coupling with maleimide-containing molecules. Unnatural amino acids were used to site-specifically incorporate (C) azide groups or (D) aldehyde groups into proteins for azide-alkyne cycloadditions or reactions with alkoxyamines respectively. Enzymatic reactions have also been employed as site-specific conjugation methods. (E) Sortase A was used to ligate functional peptides to proteins. (F) Microbial transglutaminase catalyzes the labeling of target proteins containing Q-tag sequences with lysine primary amine substrates. (G) Phosphopantetheinyl transferases modify serine residues with coenzyme A (CoA) derivatives. (H) Prenyltransferases including farnesyltransferase or geranylgeranyltransferase can be used to modify proteins with natural isoprenoids or their derivatives functionalized with reactive groups for corresponding coupling reactions.