Molecular Mechanisms of Intracellular Delivery of Nanoparticles Monitored by an Enzyme‑Induced Proximity Labeling

Achieving increasingly finely targeted drug delivery to organs,tissues,cells,and even to intracellular biomacromolecules is one of the core goals of nanomedicines.As the delivery destination is refined to cellular and subcellular targets,it is essential to explore the delivery of nanomedicines at the molecular level.However,due to the lack of technical methods,the molecular mechanism of the intracellular delivery of nanomedicines remains unclear to date.Here,we develop an enzyme-induced proximity labeling technology in nanoparticles(nano-EPL)for the real-time monitoring of proteins that interact with intracellular nanomedicines.Poly(lactic-co-glycolic acid)nanoparticles coupled with horseradish peroxidase(HRP)were fabricated as a model(HRP(+)-PNPs)to evaluate the molecular mechanism of nano delivery in macrophages.By adding the labeling probe biotin-phenol and the catalytic substrate H_(2)O_(2)at different time points in cellular delivery,nano-EPL technology was validated for the real-time in situ labeling of proteins interacting with nanoparticles.Nano-EPL achieves the dynamic molecular profiling of 740 proteins to map the intracellular delivery of HRP(+)-PNPs in macrophages over time.Based on dynamic clustering analysis of these proteins,we further discovered that different organelles,including endosomes,lysosomes,the endoplasmic reticulum,and the Golgi apparatus,are involved in delivery with distinct participation timelines.More importantly,the engagement of these organelles differentially affects the drug delivery efficiency,reflecting the spatial–temporal heterogeneity of nano delivery in cells.In summary,these findings highlight a significant methodological advance toward understanding the molecular mechanisms involved in the intracellular delivery of nanomedicines.

TurboID Proximity Labeling of a Protocadherin Protein to Characterize Interacting Protein Complex

The study of the neuron has always been a fundamental aspect when it came to studying mental illnesses such as autism and depression. The protein protocadherin-9 (PCDH9) is an important transmembrane protein in the development of the neuron synapse. Hence, research on its protein interactome is key to understanding its functionality and specific properties. A newly discovered biotin ligase, TurboID, is a proximity labeler that is designed to be able to label and observe transmembrane proteins, something that previous methods struggled with. The TurboID method is verified in HEK293T cells and primary cultured mouse cortical neurons. Results have proven the validity of the TurboID method in observing PCDH9-interacting proteins.

Proximity labeling: an emerging tool for probing in planta molecular interactions

Protein–protein interaction(PPI)networks are key to nearly all aspects of cellular activity.Therefore,the identification of PPIs is important for understanding a specific biological process in an organism.Compared with conventional methods for probing PPIs,the recently described proximity labeling(PL)approach combined with mass spectrometry(MS)-based quantitative proteomics hasemerged as apowerful approach for characterizing PPIs.However,the application of PL in planta remains in its infancy.Here,we summarize recent progress in PL and its potential utilization in plant biology.We specifically summarize advances in PL,including the development and comparison of different PL enzymes and the application of PL for deciphering various molecular interactions in different organisms with an emphasis on plant systems.

Defining Proximity Proteome of Histone Modifications by Antibody-mediated Protein A-APEX2 Labeling

Proximity labeling catalyzed by promiscuous enzymes,such as APEX2,has emerged as a powerful approach to characterize multiprotein complexes and protein-protein interactions.However,current methods depend on the expression of exogenous fusion proteins and cannot be applied to identify proteins surrounding post-translationally modified proteins.To address this limitation,we developed a new method to label proximal proteins of interest by antibody-mediated protein A-ascorbate peroxidase 2(pA-APEX2) labeling(AMAPEX).In this method,a modified protein is bound in situ by a specific antibody,which then tethers a pA-APEX2 fusion protein.Activation of APEX2 labels the nearby proteins with biotin;the biotinylated proteins are then purified using streptavidin beads and identified by mass spectrometry.We demonstrated the utility of this approach by profiling the proximal proteins of histone modifications including H3 K27 me3,H3 K9 me3,H3 K4 me3,H4 K5 ac,and H4 K12 ac,as well as verifying the co-localization of these identified proteins with bait proteins by published ChIP-seq analysis and nucleosome immunoprecipitation.Overall,AMAPEX is an efficient method to identify proteins that are proximal to modified histones.

Proximity Chemistry in Living Systems

Enzyme-and catalyst-generated reactive species have been leveraged in the past decade to covalently label biomolecules within a short range of a defined site or space inside cells or at the cell–cell interface.Due to their high spatial resolution,such proximity labeling strategies have been coupled with various bioanalytical techniques for dissecting dynamic and complex biological processes.Here,we review the development of enzyme-and catalyst-triggered proximity chemistry and their applications to identifying protein interaction networks as well as cell–cell communications in living systems.

Specific pupylation as IDEntity reporter(SPIDER)for the identification of protein-biomolecule interactions

Protein-biomolecule interactions play pivotal roles in almost all biological processes.For a biomolecule of interest,the identification of the interacting protein(s)is essential.For this need,although many assays are available,highly robust and reliable methods are always desired.By combining a substrate-based proximity labeling activity from the pupylation pathway of Mycobacterium tuberculosis and the streptavidin(SA)-biotin system,we developed the Specific Pupylation as IDEntity Reporter(SPIDER)method for identifying protein-biomolecule interactions.Using SPIDER,we validated the interactions between the known binding proteins of protein,DNA,RNA,and small molecule.We successfully applied SPIDER to construct the global protein interactome for m^(6)A and m RNA,identified a variety of uncharacterized m^(6)A binding proteins,and validated SRSF7 as a potential m^(6)A reader.We globally identified the binding proteins for lenalidomide and Cob B.Moreover,we identified SARS-CoV-2-specific receptors on the cell membrane.Overall,SPIDER is powerful and highly accessible for the study of proteinbiomolecule interactions.