No abstract

Studying posttranslational modifications classically relies on experimental strategies that oversimplify the complex biosynthetic machineries of living cells. Protein glycosylation contributes to essential biological processes, but correlating glycan structure, underlying protein, and disease-relevant biosynthetic regulation is currently elusive. Here, we engineer living cells to tag glycans with editable chemical functionalities while providing information on biosynthesis, physiological context, and glycan fine structure. We introduce a non-natural substrate biosynthetic pathway and use engineered glycosyltransferases to incorporate chemically tagged sugars into the cell surface glycome of the living cell. We apply the strategy to a particularly redundant yet disease-relevant human glycosyltransferase family, the polypeptide N-acetylgalactosaminyl transferases. This approach bestows a gain-of-chemical-functionality modification on cells, where the products of individual glycosyltransferases can be selectively characterized or manipulated to understand glycan contribution to major physiological processes.

Mitochondria are organelles with critical roles in key processes within eukaryotic cells, and their dysfunction is linked with numerous diseases including neurodegenerative disorders and cancer. Pharmacological manipulation of mitochondrial function is therefore important both for basic science research and eventually, clinical medicine. However, in comparison to other organelles, mitochondria are difficult to access due to their hydrophobic and dense double membrane system as well as their negative membrane potential. To tackle the challenge of targeting these important subcellular compartments, significant effort has been put forward to develop mitochondria-targeted systems capable of transporting bioactive cargo into the mitochondrial interior. Systems now exist that utilize small molecule, peptide, liposome, and nanoparticle-based transport. The vectors available vary in size and structure and can facilitate transport of a variety of compounds for mitochondrial delivery. Notably, peptide-based delivery scaffolds offer attractive features such as ease of synthesis, tunability, biocompatibility, and high uptake both in cellulo and in vivo. Owing to their simple and modular synthesis, these peptides are highly adaptable for delivering chemically diverse cargo. Key design features of mitochondria-targeted peptides include cationic charge, which allows them to harness the negative membrane potential of mitochondria, and lipophilicity, which permits favorable interaction with hydrophobic membranes of mitochondria. These peptides have been covalently tethered to target therapeutic agents, including anticancer drugs, to enhance their drug properties, and to provide probes for mitochondrial biology. Interestingly, mitochondria-targeted DNA damaging agents demonstrate high potency and the ability to evade resistance mechanisms and off-target effects. Moreover, a combination of mitochondria-targeted DNA damaging agents was applied to an siRNA screen for the elucidation of poorly understood mitochondrial DNA repair and replication pathways. In this work, a variety of novel proteins were identified that are essential for the maintenance of mitochondrial nucleic acids. Mitochondria-targeted peptides have also been used to increase the therapeutic window of antibacterial drugs with significant mammalian toxicity. Given the evolutionary similarity of mitochondria and bacteria, peptides are effective transporters that can target both of these entities. These antimicrobial peptides are highly effective even in difficult to target intracellular bacteria which reside within host cells. This peptide-based approach to targeting mitochondria has provided a variety of insights into the "druggability" of mitochondria and new biological processes that could be future drug targets. Nevertheless, the mitochondrial-targeting field is quite nascent and many exciting applications of organelle-specific conjugates remain to be explored. In this Account, we highlight the development and optimization of the mitochondria-penetrating peptides th...

Summary An analogue of the anticancer drug cisplatin (mtPt) was delivered to mitochondria of human cells using a peptide specifically targeting this organelle. mtPt induces apoptosis without damaging nuclear DNA, indicating that mtDNA damage is sufficient to mediate the activity of a platinum-based chemotherapeutic. This study is the first to demonstrate specific delivery of a platinum drug to mitochondria and to investigate the effects of directing this agent outside the nucleus.

Efficient and accurate replication and repair of mitochondrial DNA is essential for cellular viability, yet only a minimal complement of mitochondrial proteins with relevant activities have been identified. Here, we describe an approach to screen for new pathways involved in the maintenance of mitochondrial DNA (mtDNA) that leverages the activities of DNA-damaging probes exhibiting specific subcellular localization. By conducting a siRNA screen of known nuclear DNA maintenance factors, and monitoring synergistic effects of gene depletion on the activity of mitochondria-specific DNA-damaging agents, we identify a series of proteins not previously recognized to act within mitochondria. These include proteins that function in pathways of oxidative DNA damage repair and dsDNA break repair, along with a novel mitochondrial DNA polymerase, POLθ, that facilitates efficient DNA replication in an environment prone to oxidative stress. POLθ expression levels affect the mutational rate of mitochondrial DNA, but this protein also appears critical for efficient mtDNA replication.

Glyco-immune checkpoint receptors, molecules that inhibit immune cell activity following binding to glycosylated cell-surface antigens, are emerging as attractive targets for cancer immunotherapy. Defining biologically relevant ligands that bind and activate such receptors, however, has historically been a significant challenge. Here, we present a CRISPRi genomic screening strategy that allowed unbiased identification of the key genes required for cell-surface presentation of glycan ligands on leukemia cells that bind the glyco-immune checkpoint receptors Siglec-7 and Siglec-9. This approach revealed a selective interaction between Siglec-7 and the mucin-type glycoprotein CD43. Further work identified a specific N-terminal glycopeptide region of CD43 containing clusters of disialylated O-glycan tetrasaccharides that form specific Siglec-7 binding motifs. Knockout or blockade of CD43 in leukemia cells relieves Siglec-7-mediated inhibition of immune killing activity. This work identifies a potential target for immune checkpoint blockade therapy and represents a generalizable approach to dissection of glycan–receptor interactions in living cells.

Protein glycosylation events that happen early in the secretory pathway are often dysregulated during tumorigenesis. These events can be probed, in principle, by monosaccharides with bioorthogonal tags that would ideally be specific for distinct glycan subtypes. However, metabolic interconversion into other monosaccharides drastically reduces such specificity in the living cell. Here, we use a structure-based design process to develop the monosaccharide probe N-(S)-azidopropionylgalactosamine (GalNAzMe) that is specific for cancer-relevant Ser/Thr(O)–linked N-acetylgalactosamine (GalNAc) glycosylation. By virtue of a branched N-acylamide side chain, GalNAzMe is not interconverted by epimerization to the corresponding N-acetylglucosamine analog by the epimerase N-acetylgalactosamine–4-epimerase (GALE) like conventional GalNAc–based probes. GalNAzMe enters O-GalNAc glycosylation but does not enter other major cell surface glycan types including Asn(N)-linked glycans. We transfect cells with the engineered pyrophosphorylase mut-AGX1 to biosynthesize the nucleotide-sugar donor uridine diphosphate (UDP)-GalNAzMe from a sugar-1-phosphate precursor. Tagged with a bioorthogonal azide group, GalNAzMe serves as an O-glycan–specific reporter in superresolution microscopy, chemical glycoproteomics, a genome-wide CRISPR-knockout (CRISPR-KO) screen, and imaging of intestinal organoids. Additional ectopic expression of an engineered glycosyltransferase, “bump-and-hole” (BH)–GalNAc-T2, boosts labeling in a programmable fashion by increasing incorporation of GalNAzMe into the cell surface glycoproteome. Alleviating the need for GALE-KO cells in metabolic labeling experiments, GalNAzMe is a precision tool that allows a detailed view into the biology of a major type of cancer-relevant protein glycosylation.

The mitochondria within human cells play a major role in a variety of critical processes involved in cell survival and death. An understanding of mitochondrial involvement in various human diseases has generated an appreciable amount of interest in exploring this organelle as a potential drug target. As a result, a number of strategies to probe and combat mitochondria-associated diseases have emerged. Access to mitochondria-specific delivery vectors has allowed the study of biological processes within this intracellular compartment with a heightened level of specificity. In this review, we summarize the features of existing delivery vectors developed for targeting probes and therapeutics to this highly impermeable organelle. We also discuss the major applications of mitochondrial targeting of bioactive molecules, which include the detection and treatment of oxidative damage, combating bacterial infections, and the development of new therapeutic approaches for cancer. Future directions include the assessment of the therapeutic benefit achieved by mitochondrial targeting for treatment of disease in vivo. In addition, the availability of mitochondria-specific chemical probes will allow the elucidation of the details of biological processes that occur within this cellular compartment.