Molecular basis of SAP05-mediated ubiquitin-independent proteasomal degradation of transcription factors.

SAP05, a secreted effector by the obligate parasitic bacteria phytoplasma, bridges host SPL and GATA transcription factors (TFs) to the 26 S proteasome subunit RPN10 for ubiquitination-independent degradation. Here, we report the crystal structures of SAP05 in complex with SPL5, GATA18 and RPN10, which provide detailed insights into the protein-protein interactions involving SAP05. SAP05 employs two opposing lobes with an acidic path and a hydrophobic path to contact TFs and RPN10, respectively. Our crystal structures, in conjunction with mutagenesis and degradation assays, reveal that SAP05 targets plant GATAs but not animal GATAs dependent on their direct salt-bridged electrostatic interactions. Additionally, SAP05 hijacks plant RPN10 but not animal RPN10 due to structural steric hindrance and the key hydrophobic interactions. This study provides valuable molecular-level information into the modulation of host proteins to prevent insect-borne diseases.

Metformin rejuvenates Nap1l2-impaired immunomodulation of bone marrow mesenchymal stem cells via metabolic reprogramming.

Ageing and cell senescence of mesenchymal stem cells (MSCs) limited their immunomodulation properties and therapeutic application. We previously reported that nucleosome assembly protein 1-like 2 (Nap1l2) contributes to MSCs senescence and osteogenic differentiation. Here, we sought to evaluate whether Nap1l2 impairs the immunomodulatory properties of MSCs and find a way to rescue the deficient properties. We demonstrated that metformin could rescue the impaired migration properties and T cell regulation properties of OE-Nap1l2 BMSCs. Moreover, metformin could improve the impaired therapeutic efficacy of OE-Nap1l2 BMSCs in the treatment of colitis and experimental autoimmune encephalomyelitis in mice. Mechanistically, metformin was capable of upregulating the activation of AMPK, synthesis of l-arginine and expression of inducible nitric oxide synthase in OE-Nap1l2 BMSCs, leading to an increasing level of nitric oxide. This study indicated that Nap1l2 negatively regulated the immunomodulatory properties of BMSCs and that the impaired functions could be rescued by metformin pretreatment via metabolic reprogramming. This strategy might serve as a practical therapeutic option to rescue impaired MSCs functions for further application.

Glutathione-responsive PROTAC for targeted degradation of ERα in breast cancer cells.

ERα (estrogen receptor-α)-targeting PROTACs (PROteolysis TArgeting Chimeras) have emerged as a novel and promising modality for breast cancer therapeutics. However, ERα PROTACs-induced degradation in normal tissues raises concerns about potential off-tissue toxicity. Tumor microenvironment-responsive strategy provides potential for specific control of the PROTAC's on-target degradation activity. The glutathione (GSH) level has been reported to be significantly increased in tumor cells. Here, we designed a GSH-responsive ERα PROTAC, which is generated by conjugating an o-nitrobenzenesulfonyl group to the hydroxyl group of VHL-based ERα PROTAC through a nucleophilic substitution reaction. The o-nitrobenzenesulfonyl group as a protecting group blocks the bioactivity of ERα PROTAC (ER-P1), and that can be specifically recognized and removed by highly abundant GSH in cancer cells. Consequently, the GSH-responsive ERα PROTAC (GSH-ER-P1) exhibits significantly enhanced degradation of ERα in cancer cells compared to that in normal cells, leading to a remarkable inhibition of breast cancer cell proliferation and less toxic effects on normal cells. This study provides a potentially valuable strategy for breast cancer treatment using tumor microenvironment-responsive PROTACs.

Selective degradation of BRD4 suppresses lung cancer cell proliferation using GSH-responsive PROTAC precursors.

BRD4,as a transcriptional and epigenetic regulator to mediate cellular functions, plays an important role in cancer development.Targeting BRD4 with conventional inhibitors in cancer therapy requires high doses, which often leads to off-target and adverse effects. BRD4-targeted proteolysis-targeting chimeras (PROTACs) can catalytically degrade BRD4 utilizing the endogenous proteasome system, and exhibit promising anti-tumor activity. However, most of the developed PROTACs are non-cancer specific and relatively toxic towards normal cells, limiting their practical applications in cancer treatment. By taking advantage of higher glutathione (GSH) levels in cancer cells than that in normal cells, we developed several GSH-responsive PROTAC precursors 1a-c via the attachment of a GSH-trigger unit on the hydroxyl group of the VHL (von Hippel-Lindau) ligand for the recruitment of E3 ligase. Among the precursors, 1a can be efficiently activated by the innately higher concentrations of GSH in lung cancer cells (A549 and H1299) to release active PROTAC 1, degrading intracellular BRD4 and resulting in cytotoxicity, which is confirmed by mechanistic investigation. On the other hand, 1a cannot be efficiently triggered in normal lung cells (WI38 and HULEC-5a) containing lower levels of GSH, therefore reducing the adverse effects on normal cells. This work provides an alternative proof of concept approach for developing stimuli-responsive PROTAC precursors, and affords a novel insight to improve the selectivity and minimize the adverse effects of current PROTACs, hence enhancing their clinical potential.

Hydrogen Peroxide-Inducible PROTACs for Targeted Protein Degradation in Cancer Cells.

Proteolysis-targeting chimeras (PROTACs) provide a powerful technique to degrade targeted proteins utilizing the cellular ubiquitin-proteasome system. The major concern is the host toxicity resulting from their poor selectivity. Inducible PROTACs responding to exogenous stimulus, such as light, improve their specificity, but it is difficult for photo-activation in deep tissues. Herein, we develop H2 O2 -inducible PROTAC precursors 2/5, which can be activated by endogenous H2 O2 in cancer cells to release the active PROTACs 1/4 to effectively degrade targeted proteins. This results in the intended cytotoxicity towards cancer cells while targeted protein in normal cells remains almost unaffected. The higher Bromodomain-containing protein 4 (BRD4) degradation activity and cytotoxicity of 2 towards cancer cells is mainly due to the higher endogenous concentration of H2 O2 in cancer cells (A549 and H1299), characterized by H2 O2 -responsive fluorescence probe 3. Western blot assays and cytotoxicity experiments demonstrate that 2 degrades BRD4 more effectively and is more cytotoxic in H2 O2 -rich cancer cells than in H2 O2 -deficient normal cells. This method is also extended to estrogen receptor (ER)-PROTAC precursor 5, showing H2 O2 -dependent ER degradation ability. Thus, we establish a novel strategy to induce targeted protein degradation in a H2 O2 -dependent way, which has the potential to improve the selectivity of PROTACs.

Recognition of an Ala-rich C-degron by the E3 ligase Pirh2.

The ribosome-associated quality-control (RQC) pathway degrades aberrant nascent polypeptides arising from ribosome stalling during translation. In mammals, the E3 ligase Pirh2 mediates the degradation of aberrant nascent polypeptides by targeting the C-terminal polyalanine degrons (polyAla/C-degrons). Here, we present the crystal structure of Pirh2 bound to the polyAla/C-degron, which shows that the N-terminal domain and the RING domain of Pirh2 form a narrow groove encapsulating the alanine residues of the polyAla/C-degron. Affinity measurements in vitro and global protein stability assays in cells further demonstrate that Pirh2 recognizes a C-terminal A/S-X-A-A motif for substrate degradation. Taken together, our study provides the molecular basis underlying polyAla/C-degron recognition by Pirh2 and expands the substrate recognition spectrum of Pirh2.

CRL2ZER1/ZYG11B recognizes small N-terminal residues for degradation.

N-degron pathway plays an important role in the protein quality control and maintenance of cellular protein homeostasis. ZER1 and ZYG11B, the substrate receptors of the Cullin 2-RING E3 ubiquitin ligase (CRL2), recognize N-terminal (Nt) glycine degrons and participate in the Nt-myristoylation quality control through the Gly/N-degron pathway. Here we show that ZER1 and ZYG11B can also recognize small Nt-residues other than glycine. Specifically, ZER1 binds better to Nt-Ser, -Ala, -Thr and -Cys than to -Gly, while ZYG11B prefers Nt-Gly but also has the capacity to recognize Nt-Ser, -Ala and -Cys in vitro. We found that Nt-Ser, -Ala and -Cys undergo Nt-acetylation catalyzed by Nt-acetyltransferase (NAT), thereby shielding them from recognition by ZER1/ZYG11B in cells. Instead, ZER1/ZYG11B readily targets a selection of small Nt-residues lacking Nt-acetylation for degradation in NAT-deficient cells, implicating its role in the Nt-acetylation quality control. Furthermore, we present the crystal structures of ZER1 and ZYG11B bound to various small Nt-residues and uncover the molecular mechanism of non-acetylated substrate recognition by ZER1 and ZYG11B.

C-terminal glutamine acts as a C-degron targeted by E3 ubiquitin ligase TRIM7.

The exposed N-terminal or C-terminal residues of proteins can act, in cognate sequence contexts, as degradation signals (degrons) that are targeted by specific E3 ubiquitin ligases for proteasome-dependent degradation by N-degron or C-degron pathways. Here, we discovered a distinct C-degron pathway, termed the Gln/C-degron pathway, in which the B30.2 domain of E3 ubiquitin ligase TRIM7 (TRIM7B30.2) mediates the recognition of proteins bearing a C-terminal glutamine. By determining crystal structures of TRIM7B30.2 in complexes with various peptides, we show that TRIM7B30.2 forms a positively charged binding pocket to engage the "U"-shaped Gln/C-degron. The four C-terminal residues of a substrate play an important role in C-degron recognition, with C-terminal glutamine as the principal determinant. In vitro biochemical and cellular experiments were used to further analyze the substrate specificity and selective degradation of the Gln/C-degron by TRIM7.

Selective Antitumor Activity and Photocytotoxicity of Glutathione-Activated Abasic Site Trapping Agents.

Abasic (AP) sites are one of the most common DNA lesions in cells. Aldehyde-reactive alkoxyamines capture AP sites and block the activity of APE1, the enzyme responsible for initiating their repair. Blocking the APE1 repair of AP sites leads to cell death, and it is an actively investigated approach for treating cancer. However, unselective AP site capture in different cells produces side effects and limits the application of alkoxyamines in chemotherapy. Herein we take advantage of the higher glutathione (GSH) concentration in cancer cells over normal cells to develop GSH-inducible agents that selectively kill cancer cells. 2,4-Dinitrobenzenesulfonamide caged coumarin-based alkoxyamines 1 and 2 are selectively revealed by GSH to release SO2 and fluorescent coumarin-based alkoxyamines 3 and 4 that trap AP sites in cells. GSH-directed AP site trapping and SO2 release result in selective cytotoxicity (defined as IC50WI38/IC50H1299) against H1299 lung cancer cells over normal WI38 lung cells, ranging from 1.8 to 2.8 for 1 and 2. The alkylating agent methylmethanesulfonate (MMS) promotes the formation of AP sites in cells and enhances the cytotoxicity of agent 1 in a dose-dependent way. Moreover, the comet assay and γH2AX assay suggest that AP adducts form a highly toxic DNA interstrand cross-link (ICL) upon photolysis, leading to further cell death. DNA flow cytometric analysis showed that 1 promoted cell apoptosis in the early stage and induced G2/M phase cell-cycle arrest. The 2,4-dinitrobenzenesulfonamide-caged alkoxyamines exhibited selective antitumor activity and photocytotoxicity in cancer cells, illuminating their potential as GSH-directed chemotherapeutic agents.

Molecular basis for recognition of Gly/N-degrons by CRL2ZYG11B and CRL2ZER1.

N-degron pathways are a set of proteolytic systems that target the N-terminal destabilizing residues of substrates for proteasomal degradation. Recently, the Gly/N-degron pathway has been identified as a new branch of the N-degron pathway. The N-terminal glycine degron (Gly/N-degron) is recognized by ZYG11B and ZER1, the substrate receptors of the Cullin 2-RING E3 ubiquitin ligase (CRL2). Here we present the crystal structures of ZYG11B and ZER1 bound to various Gly/N-degrons. The structures reveal that ZYG11B and ZER1 utilize their armadillo (ARM) repeats forming a deep and narrow cavity to engage mainly the first four residues of Gly/N-degrons. The α-amino group of the Gly/N-degron is accommodated in an acidic pocket by five conserved hydrogen bonds. These structures, together with biochemical studies, decipher the molecular basis for the specific recognition of the Gly/N-degron by ZYG11B and ZER1, providing key information for future structure-based chemical probe design.

Molecular basis for ubiquitin ligase CRL2FEM1C-mediated recognition of C-degron.

Proteome integrity depends on the ubiquitin-proteasome system to degrade unwanted or abnormal proteins. In addition to the N-degrons, C-terminal residues of proteins can also serve as degradation signals (C-degrons) that are recognized by specific cullin-RING ubiquitin ligases (CRLs) for proteasomal degradation. FEM1C is a CRL2 substrate receptor that targets the C-terminal arginine degron (Arg/C-degron), but the molecular mechanism of substrate recognition remains largely elusive. Here, we present crystal structures of FEM1C in complex with Arg/C-degron and show that FEM1C utilizes a semi-open binding pocket to capture the C-terminal arginine and that the extreme C-terminal arginine is the major structural determinant in recognition by FEM1C. Together with biochemical and mutagenesis studies, we provide a framework for understanding molecular recognition of the Arg/C-degron by the FEM family of proteins.

Histone H3K23-specific acetylation by MORF is coupled to H3K14 acylation.

Acetylation of histone H3K23 has emerged as an essential posttranslational modification associated with cancer and learning and memory impairment, yet our understanding of this epigenetic mark remains insufficient. Here, we identify the native MORF complex as a histone H3K23-specific acetyltransferase and elucidate its mechanism of action. The acetyltransferase function of the catalytic MORF subunit is positively regulated by the DPF domain of MORF (MORFDPF). The crystal structure of MORFDPF in complex with crotonylated H3K14 peptide provides mechanistic insight into selectivity of this epigenetic reader and its ability to recognize both histone and DNA. ChIP data reveal the role of MORFDPF in MORF-dependent H3K23 acetylation of target genes. Mass spectrometry, biochemical and genomic analyses show co-existence of the H3K23ac and H3K14ac modifications in vitro and co-occupancy of the MORF complex, H3K23ac, and H3K14ac at specific loci in vivo. Our findings suggest a model in which interaction of MORFDPF with acylated H3K14 promotes acetylation of H3K23 by the native MORF complex to activate transcription.

The ZZ domain as a new epigenetic reader and a degradation signal sensor.

Although relatively small in size, the ZZ-type zinc finger (ZZ) domain is a versatile signaling module that is implicated in a diverse set of cell signaling events. Here, we highlight the most recent studies focused on the ZZ domain function as a histone reader and a sensor of protein degradation signals. We review and compare the molecular and structural mechanisms underlying targeting the amino-terminal sequences of histone H3 and arginylated substrates by the ZZ domain. We also discuss the ZZ domain sensitivity to histone PTMs and summarize biological outcomes associated with the recognition of histone and non-histone ligands by the ZZ domain-containing proteins and complexes.

Structural insights into the π-π-π stacking mechanism and DNA-binding activity of the YEATS domain.

The YEATS domain has been identified as a reader of histone acylation and more recently emerged as a promising anti-cancer therapeutic target. Here, we detail the structural mechanisms for π-π-π stacking involving the YEATS domains of yeast Taf14 and human AF9 and acylated histone H3 peptides and explore DNA-binding activities of these domains. Taf14-YEATS selects for crotonyllysine, forming π stacking with both the crotonyl amide and the alkene moiety, whereas AF9-YEATS exhibits comparable affinities to saturated and unsaturated acyllysines, engaging them through π stacking with the acyl amide. Importantly, AF9-YEATS is capable of binding to DNA, whereas Taf14-YEATS is not. Using a structure-guided approach, we engineered a mutant of Taf14-YEATS that engages crotonyllysine through the aromatic-aliphatic-aromatic π stacking and shows high selectivity for the crotonyl H3K9 modification. Our findings shed light on the molecular principles underlying recognition of acyllysine marks and reveal a previously unidentified DNA-binding activity of AF9-YEATS.

ZZ-dependent regulation of p62/SQSTM1 in autophagy.

Autophagic receptor p62 is a critical mediator of cell detoxification, stress response, and metabolic programs and is commonly deregulated in human diseases. The diverse functions of p62 arise from its ability to interact with a large set of ligands, such as arginylated (Nt-R) substrates. Here, we describe the structural mechanism for selective recognition of Nt-R by the ZZ domain of p62 (p62ZZ). We show that binding of p62ZZ to Nt-R substrates stimulates p62 aggregation and macroautophagy and is required for autophagic targeting of p62. p62 is essential for mTORC1 activation in response to arginine, but it is not a direct sensor of free arginine in the mTORC1 pathway. We identified a regulatory linker (RL) region in p62 that binds p62ZZ in vitro and may modulate p62 function. Our findings shed new light on the mechanistic and functional significance of the major cytosolic adaptor protein p62 in two fundamental signaling pathways.

The ZZ-type zinc finger of ZZZ3 modulates the ATAC complex-mediated histone acetylation and gene activation.

Recognition of histones by epigenetic readers is a fundamental mechanism for the regulation of chromatin and transcription. Most reader modules target specific post-translational modifications on histones. Here, we report the identification of a reader of histone H3, the ZZ-type zinc finger (ZZ) domain of ZZZ3, a subunit of the Ada-two-A-containing (ATAC) histone acetyltransferase complex. The solution NMR structure of the ZZ in complex with the H3 peptide reveals a unique binding mechanism involving caging of the N-terminal Alanine 1 of histone H3 in an acidic cavity of the ZZ domain, indicating a specific recognition of H3 versus other histones. Depletion of ZZZ3 or disruption of the ZZ-H3 interaction dampens ATAC-dependent promoter histone H3K9 acetylation and target gene expression. Overall, our study identifies the ZZ domain of ZZZ3 as a histone H3 reader that is required for the ATAC complex-mediated maintenance of histone acetylation and gene activation.

The ZZ domain of p300 mediates specificity of the adjacent HAT domain for histone H3.

Human p300 is a transcriptional co-activator and a major acetyltransferase that acetylates histones and other proteins facilitating gene transcription. The activity of p300 relies on the fine-tuned interactome that involves a dozen p300 domains and hundreds of binding partners and links p300 to a wide range of vital signaling events. Here, we report a novel function of the ZZ-type zinc finger (ZZ) of p300 as a reader of histone H3. We show that the ZZ domain and acetyllysine-recognizing bromodomain of p300 play critical roles in modulating p300 enzymatic activity and its association with chromatin. The acetyllysine binding function of bromodomain is essential for acetylation of histones H3 and H4, whereas interaction of the ZZ domain with H3 promotes selective acetylation of the histone H3K27 and H3K18 sites.

BMI1 regulates androgen receptor in prostate cancer independently of the polycomb repressive complex 1.

BMI1, a polycomb group (PcG) protein, plays a critical role in epigenetic regulation of cell differentiation and proliferation, and cancer stem cell self-renewal. BMI1 is upregulated in multiple types of cancer, including prostate cancer. As a key component of polycomb repressive complex 1 (PRC1), BMI1 exerts its oncogenic functions by enhancing the enzymatic activities of RING1B to ubiquitinate histone H2A at lysine 119 and repress gene transcription. Here, we report a PRC1-independent role of BMI1 that is critical for castration-resistant prostate cancer (CRPC) progression. BMI1 binds the androgen receptor (AR) and prevents MDM2-mediated AR protein degradation, resulting in sustained AR signaling in prostate cancer cells. More importantly, we demonstrate that targeting BMI1 effectively inhibits tumor growth of xenografts that have developed resistance to surgical castration and enzalutamide treatment. These results suggest that blocking BMI1 alone or in combination with anti-AR therapy can be more efficient to suppress prostate tumor growth.

O-GlcNAcylation of SIRT1 enhances its deacetylase activity and promotes cytoprotection under stress.

SIRT1 is the most evolutionarily conserved mammalian sirtuin, and it plays a vital role in the regulation of metabolism, stress responses, genome stability, and ageing. As a stress sensor, SIRT1 deacetylase activity is significantly increased during stresses, but the molecular mechanisms are not yet fully clear. Here, we show that SIRT1 is dynamically modified with O-GlcNAc at Ser 549 in its carboxy-terminal region, which directly increases its deacetylase activity both in vitro and in vivo. The O-GlcNAcylation of SIRT1 is elevated during genotoxic, oxidative, and metabolic stress stimuli in cellular and mouse models, thereby increasing SIRT1 deacetylase activity and protecting cells from stress-induced apoptosis. Our findings demonstrate a new mechanism for the activation of SIRT1 under stress conditions and suggest a novel potential therapeutic target for preventing age-related diseases and extending healthspan.

YEATS2 links histone acetylation to tumorigenesis of non-small cell lung cancer.

Recognition of modified histones by "reader" proteins constitutes a key mechanism regulating diverse chromatin-associated processes important for normal and neoplastic development. We recently identified the YEATS domain as a novel acetyllysine-binding module; however, the functional importance of YEATS domain-containing proteins in human cancer remains largely unknown. Here, we show that the YEATS2 gene is highly amplified in human non-small cell lung cancer (NSCLC) and is required for cancer cell growth and survival. YEATS2 binds to acetylated histone H3 via its YEATS domain. The YEATS2-containing ATAC complex co-localizes with H3K27 acetylation (H3K27ac) on the promoters of actively transcribed genes. Depletion of YEATS2 or disruption of the interaction between its YEATS domain and acetylated histones reduces the ATAC complex-dependent promoter H3K9ac levels and deactivates the expression of essential genes. Taken together, our study identifies YEATS2 as a histone H3K27ac reader that regulates a transcriptional program essential for NSCLC tumorigenesis.