Nature chemical biology最新文献

The physical properties of cellular membranes are influenced by protein and lipid interactions. In situ proximity labeling interactomic methods are well suited to characterize these dynamic and often fleeting interactions. Yet, available methods require distinct chemistries for proteins and lipids. Here we establish a singlet oxygen-based photocatalytic proximity labeling platform (POCA) that reports intracellular interactomes for both proteins and lipids using cell-penetrant photosensitizer reagents. Cholesterol-directed POCA captured known and unprecedented cholesterol-binding proteins, including protein complexes sensitive to intracellular cholesterol levels and proteins uniquely captured by physiologically relevant lipoprotein uptake. Protein-directed POCA accurately mapped intracellular membrane complexes, defined sterol-dependent changes to the interactome of the cholesterol transport protein Aster-B and revealed singlet oxygen-mediated domain-specific Aster crosslinking. More broadly, we find that POCA is a versatile interactomics platform that is straightforward to implement, using the readily available HaloTag system, fulfilling unmet needs in intracellular singlet oxygen-based proximity labeling proteomics.

Molecules that facilitate protein-protein interactions are immensely impactful. However, such compounds typically rely on accessory proteins to function, such as E3 ligases for targeted degradation, which may restrict their scope or lead to resistance. We alleviate the need for accessory proteins with a strategy that exploits protein symmetry as a selective vulnerability and is widely applicable because of the ubiquitous nature of homomeric proteins. We target homomeric proteins with PINCHs (polymerization-inducing chimeras)-bifunctional molecules composed of two linked ligands that bridge homomers and trigger their supramolecular assembly into insoluble polymers. We design PINCHs that achieve efficient polymerization of four targets. In cells, we observed that a PINCH targeting Keap1 exhibited a longer duration of action and a PINCH targeting BCL6 displayed selective lowering of B cell viability compared to their monomeric parents. Our results highlight PINCHs as a novel and general strategy to modulate and knock out protein function.

CRISPR-Cas9, an RNA-guided immune system, functions specifically in bacteria while controlling autoimmunity. However, its application to genome editing often causes deleterious off-target cleavages. Here, by sequencing CRISPR RNAs (crRNAs), we discovered abasic modifications that naturally suppress off-target self-cleavages from activated Cas9 in Streptococcus pyogenes (SpCas9). Bacteriophage infection induces oxidative stress, preferentially oxidizing the 5' end of crRNAs into abasic modifications. Mechanistically, abasic substitutions at the 5' end reduce off-target effects by limiting base pairing while preserving SpCas9-interacting backbones to maintain on-target efficiency. Abasic extensions at the 5' end reduce off-target effects by sterically constraining SpCas9 but retain on-target activity by avoiding extra base pairs. Moreover, these approaches can be combined (abasic substitution and extension), enhancing SpCas9 fidelity by increasing mismatch intolerance at the protospacer-adjacent motif-distal region and outperforming SpCas9 variants. Biologically inspired, we developed abasic chemical modifications for guide RNAs that improve CRISPR-Cas9 genome-editing specificity, demonstrating potential for in vivo application.