Cheng Dong

The Gly/N-degron pathway is a branch of the proteasomal degradation pathway that specifically targets proteins initiated with an N-terminal glycine. The E3 ligase substrate adaptors ZYG11B and ZER1 have been identified as being responsible for recognizing the target proteins of the Gly/N-degron pathway. Previously, it has been shown that the Gly/N-degron pathway activates the human NLRP1 inflammasome by degrading the autoinhibitory N-terminal fragment of NLRP1 after cleavage by the enteroviral 3C protease. However, the recognition of the NLRP1 Gly/N-degron is not yet fully understood. Here, we determined the X-ray crystal structure of ZER1 bound to the NLRP1 Gly/N-degron at a resolution of 2.2 Å. The structural information revealed that ZER1 uses its ARM repeats to form a conserved cavity that engages the N-terminal glycine (G1) through hydrogen bonds to Asp556, Asn597 and Glu600. Structural comparisons show a shared recognition mode for Gly/N-degrons despite subtle differences in side-chain interactions. However, ZER1 exhibits weaker affinity for the NLRP1 Gly/N-degron than ZYG11B, likely due to distinct local environments surrounding position 3. This study elucidates the molecular basis of NLRP1 recognition by ZER1 and provides insights into targeting this pathway in inflammatory diseases.

BACKGROUND: Pulmonary interstitial macrophages can be divided into 2 distinct subsets with different origins: resident macrophages (resMФs) and recruited macrophages (recMФs). However, their specific roles in pulmonary arterial hypertension remain unclear. METHODS: Bone marrow transplantation, the DT (diphtheria toxin) receptor system, and genetically modified murine models were utilized to explore how key TFs (transcription factors) regulate phenotype alterations in pulmonary resMФs and recMФs in an SU5416/hypoxia murine model of pulmonary hypertension (PH). Therapeutic approaches included DNA aptamer–based proteolysis-targeting chimera and small interfering RNA–loaded lipid nanoparticle for treating SU5416/hypoxia–exposed rats. RESULTS: Depletion of either resMФs or recMФs using DT treatment significantly reduced SU5416/hypoxia–induced PH in mice. Pulmonary recMФs exhibited a proinflammatory phenotype during PH, driven by the TF Hic1 (hypermethylated in cancer 1). Bone marrow transplantation with Hic1 ^−/− recMФs ameliorated PH in mice. Hic1 enhanced proinflammatory gene transcription by inhibiting Sirt1 (sirtuin 1)-mediated H3K9ac (histone H3 lysine 9 acetylation) deacetylation in the promoter regions. In contrast, pulmonary resMФs demonstrated a profibrotic transcriptome characterized by upregulation of MMP genes that are, in turn, regulated by Prrx2 (paired-related homeobox 2). Prrx2 deletion in resMФs protected against PH in mice by reducing perivascular fibrosis. Simultaneously targeting Prrx2 and Hic1 in macrophages significantly alleviated SU5416/hypoxia–induced PH in rats. CONCLUSIONS: The differential roles of pulmonary resMФs and recMФs in pulmonary vascular remodeling highlight novel therapeutic targets for pulmonary arterial hypertension treatment, specifically through inhibition of Hic1 and Prrx2 in macrophages.

Proteolysis targeting chimeras (PROTACs) hijack E3 ligases and the ubiquitin–proteasome system to achieve selective degradation of neo-substrates. Their ability to target otherwise intractable substrates has rendered them a valuable modality in drug discovery. However, only a handful of over 600 human E3 ligases have been functionalized for PROTAC applications. Here we show that the E3 ligase GID4 (glucose-induced degradation deficient complex 4) can be leveraged for targeted protein degradation using a noncovalent small molecule. We design and synthesize GID4-based PROTACs, exemplified by NEP162, which can eliminate endogenous BRD4 in a GID4- and ubiquitin–proteasome system-dependent manner. NEP162 exhibits antiproliferative activity and inhibits tumor growth in a xenograft model, hinting toward potential anticancer applications. We further present the crystal structures of GID4–PROTAC–BRD4 ternary complexes in three distinct states, unveiling plastic interactions between GID4 and BRD4. These structural insights, combined with in vitro and in vivo data, decipher the molecular basis by which the hereby developed PROTACs recruit BRD4 to GID4 for targeted degradation and expand our arsenal of PROTAC-exploitable E3 ligases.

As a crucial element of proteolysis targeting chimeras (PROTACs), the choice of E3 ubiquitin ligase significantly influences degradation efficacy and selectivity. However, the available arsenal of E3 ligases for PROTAC development remains underexplored, severely limiting the scope of targeted protein degradation. In this study, we identify a non-inhibitory aptamer targeting ZYG11B, a substrate receptor of the Cullin 2-RING ligase complex, as an E3 warhead for targeted protein degradation. This aptamer-based PROTAC platform, termed ZATAC, is facilely produced through bioorthogonal chemistry or self-assembly and shows promise in eliminating several undruggable target proteins, including nucleolin (NCL), SRY-box transcription factor 2 (SOX2), and mutant p53-R175H, underscoring its universality and versatility. To specifically deliver ZATACs into cancer cells, we further develop DNA three-way junction-based ZATACs (3WJ-ZATACs) by integrating an additional aptamer that selectively recognizes the protein overexpressed on the surface of cancer cells. The 3WJ-ZATACs demonstrate in vivo tumor-specific distribution and achieve dual-target degradation, thereby suppressing tumor growth without causing noticeable toxicity. In summary, ZATACs represent a general, modular, and straightforward platform for targeted protein degradation, offering insights into the potential of other untapped E3 ligases.

The ankyrin (ANK) SOCS box (ASB) family, encompassing ASB1–18, is the largest group of substrate receptors of cullin 5 Ring E3 ubiquitin ligase. Nonetheless, the mechanism of substrate recognition by ASB family proteins has remained largely elusive. Here we present the crystal structure of ASB7-Elongin B-Elongin C ternary complex bound to a conserved helical degron. ASB7 employs its ANK3-6 to form an extended groove, effectively interacting with the internal α-helix-degron through a network of side-chain-mediated electrostatic and hydrophobic interactions. Our structural findings, combined with biochemical and cellular analyses, identify the key residues of the degron motif and ASB7 required for their recognition. This will facilitate the identification of additional physiological substrates of ASB7 by providing a defined degron motif for screening. Furthermore, the structural insights provide a basis for the rational design of compounds that can specifically target ASB7 by disrupting its interaction with its cognate degron.

The C-terminal to LisH (CTLH) complex is a ubiquitin ligase complex that recognizes substrates with Pro/N-degrons via its substrate receptor Glucose-Induced Degradation 4 (GID4), but its function and substrates in humans remain unclear. Here, we report PFI-7, a potent, selective and cell-active chemical probe that antagonizes Pro/N-degron binding to human GID4. Use of PFI-7 in proximity-dependent biotinylation and quantitative proteomics enabled the identification of GID4 interactors and GID4-regulated proteins. GID4 interactors are enriched for nucleolar proteins, including the Pro/N-degron-containing RNA helicases DDX21 and DDX50. We also identified a distinct subset of proteins whose cellular levels are regulated by GID4 including HMGCS1, a Pro/N-degron-containing metabolic enzyme. These data reveal human GID4 Pro/N-degron targets regulated through a combination of degradative and nondegradative functions. Going forward, PFI-7 will be a valuable research tool for investigating CTLH complex biology and facilitating development of targeted protein degradation strategies that highjack CTLH E3 ligase activity.

A chemical probe (PFI-7) for the Gid4 subunit of the human E3 ligase CTLH degradation complex.