Cellular uptake of extracellular nucleosomes induces innate immune responses by binding and activating cGMP-AMP synthase (cGAS).

The nucleosome is the basic structural repeating unit of chromatin. DNA damage and cell apoptosis release nucleosomes into the blood circulatory system, and increased levels of circulating nucleosomes have been observed to be related to inflammation and autoimmune diseases. However, how circulating nucleosomes trigger immune responses has not been fully elucidated. cGAS (cGMP-AMP synthase) is a recently discovered pattern recognition receptor that senses cytoplasmic double-stranded DNA (dsDNA). In this study, we employed in vitro reconstituted nucleosomes to examine whether extracellular nucleosomes can gain access to the cytoplasm of mammalian cells to induce immune responses by activating cGAS. We showed that nucleosomes can be taken up by various mammalian cells. Additionally, we found that in vitro reconstituted mononucleosomes and oligonucleosomes can be recognized by cGAS. Compared to dsDNA, nucleosomes exhibit higher binding affinities to cGAS but considerably lower potency in cGAS activation. Incubation of monocytic cells with reconstituted nucleosomes leads to limited production of type I interferons and proinflammatory cytokines via a cGAS-dependent mechanism. This proof-of-concept study reveals the cGAS-dependent immunogenicity of nucleosomes and highlights the potential roles of circulating nucleosomes in autoimmune diseases, inflammation, and antitumour immunity.

Cysteine-specific protein multi-functionalization and disulfide bridging using 3-bromo-5-methylene pyrrolones.

Many reagents have been developed for cysteine-specific protein modification. However, few of them allow for multi-functionalization of a single Cys residue and disulfide bridging bioconjugation. Herein, we report 3-bromo-5-methylene pyrrolones (3Br-5MPs) as a simple, robust, and versatile class of reagents for cysteine-specific protein modification. These compounds can be facilely synthesized via a one-pot mild reaction and they show comparable tagging efficiency but higher cysteine specificity than the maleimide counterparts. The addition of cysteine to 3Br-5MPs generates conjugates that are amenable to secondary addition by another thiol or cysteine, making 3Br-5MPs valuable for multi-functionalization of a single cysteine and disulfide bridging bioconjugation. The labeling reaction and subsequent treatments are mild enough to produce stable and active protein conjugates for biological applications.

Basis for metabolite-dependent Cullin-RING ligase deneddylation by the COP9 signalosome.

The Cullin-RING ligases (CRLs) are the largest family of ubiquitin E3s activated by neddylation and regulated by the deneddylase COP9 signalosome (CSN). The inositol polyphosphate metabolites promote the formation of CRL-CSN complexes, but with unclear mechanism of action. Here, we provide structural and genetic evidence supporting inositol hexakisphosphate (IP6) as a general CSN cofactor recruiting CRLs. We determined the crystal structure of IP6 in complex with CSN subunit 2 (CSN2), based on which we identified the IP6-corresponding electron density in the cryoelectron microscopy map of a CRL4A-CSN complex. IP6 binds to a cognate pocket formed by conserved lysine residues from CSN2 and Rbx1/Roc1, thereby strengthening CRL-CSN interactions to dislodge the E2 CDC34/UBE2R from CRL and to promote CRL deneddylation. IP6 binding-deficient Csn2K70E/K70E knockin mice are embryonic lethal. The same mutation disabled Schizosaccharomyces pombe Csn2 from rescuing UV-hypersensitivity of csn2-null yeast. These data suggest that CRL transition from the E2-bound active state to the CSN-bound sequestered state is critically assisted by an interfacial IP6 small molecule, whose metabolism may be coupled to CRL-CSN complex dynamics.

A light-responsive, self-immolative linker for controlled drug delivery via peptide- and protein-drug conjugates.

When designing prodrugs, choosing an appropriate linker is the key to achieving efficient, controlled drug delivery. Herein, we report the use of a photocaged C4'-oxidized abasic site (PC4AP) as a light-responsive, self-immolative linker. Any amine- or hydroxyl-bearing drug can be loaded onto the linker via a carbamate or carbonate bond, and the linker is then conjugated to a carrier peptide or protein via an alkyl chain. The PC4AP linker is stable under physiologically relevant conditions. However, photodecaging of the linker generates an active intermediate that reacts intramolecularly with a primary amine (the ε-amine of a lysine residue and the N-terminal amine) on the carrier, leading to rapid and efficient release of the drug via an addition-elimination cascade, without generating any toxic side products. We demonstrated that the use of this self-immolative linker to conjugate the anticancer drug doxorubicin to a cell-penetrating peptide or an antibody enabled targeted, controlled delivery of the drug to cells. Our results suggest that the linker can be used with a broad range of carriers, such as cell-penetrating peptides, proteins, antibodies, and amine-functionalized polymers, and thus will find a wide range of practical applications.

4'-C-Trifluoromethyl modified oligodeoxynucleotides: synthesis, biochemical studies, and cellular uptake properties.

Herein, we report the synthesis of 4'-C-trifluoromethyl (4'-CF3) thymidine (T4'-CF3) and its incorporation into oligodeoxynucleotides (ODNs) through solid-supported DNA synthesis. The 4'-CF3 modification leads to a marginal effect on the deoxyribose conformation and a local helical structure perturbation for ODN/RNA duplexes. This type of modification slightly decreases the thermal stability of ODN/RNA duplexes (-1 °C/modification) and leads to improved nuclease resistance. Like the well-known phosphorothioate (PS) modification, heavy 4'-CF3 modifications enable direct cellular uptake of the modified ODNs without any delivery reagents. This work highlights that 4'-CF3 modified ODNs are promising candidates for antisense-based therapeutics, which will, in turn, inspire us to develop more potent modifications for antisense ODNs and siRNAs.