Naixia Zhang

Proteasome subunit Rpn13 is a novel ubiquitin receptor

Proteasomal receptors that recognize ubiquitin chains attached to substrates are key mediators of selective protein degradation in eukaryotes. Here we report the identification of a new ubiquitin receptor, Rpn13/ARM1, a known component of the proteasome. Rpn13 binds ubiquitin through a conserved amino-terminal region termed the pleckstrin-like receptor for ubiquitin (Pru) domain, which binds K48-linked diubiquitin with an affinity of approximately 90 nM. Like proteasomal ubiquitin receptor Rpn10/S5a, Rpn13 also binds ubiquitin-like (UBL) domains of UBL-ubiquitin-associated (UBA) proteins. In yeast, a synthetic phenotype results when specific mutations of the ubiquitin binding sites of Rpn10 and Rpn13 are combined, indicating functional linkage between these ubiquitin receptors. Because Rpn13 is also the proteasomal receptor for Uch37, a deubiquitinating enzyme, our findings suggest a coupling of chain recognition and disassembly at the proteasome.

Ubiquitin docking at the proteasome through a novel pleckstrin-homology domain interaction

Targeted protein degradation is largely performed by the ubiquitin–proteasome pathway, in which substrate proteins are marked by covalently attached ubiquitin chains that mediate recognition by the proteasome. It is currently unclear how the proteasome recognizes its substrates, as the only established ubiquitin receptor intrinsic to the proteasome is Rpn10/S5a (ref. 1), which is not essential for ubiquitin-mediated protein degradation in budding yeast. In the accompanying manuscript we report that Rpn13 (refs 3–7), a component of the nine-subunit proteasome base, functions as a ubiquitin receptor, complementing its known role in docking de-ubiquitinating enzyme Uch37/UCHL5 (refs 4–6) to the proteasome. Here we merge crystallography and NMR data to describe the ubiquitin-binding mechanism of Rpn13. We determine the structure of Rpn13 alone and complexed with ubiquitin. The co-complex reveals a novel ubiquitin-binding mode in which loops rather than secondary structural elements are used to capture ubiquitin. Further support for the role of Rpn13 as a proteasomal ubiquitin receptor is demonstrated by its ability to bind ubiquitin and proteasome subunit Rpn2/S1 simultaneously. Finally, we provide a model structure of Rpn13 complexed to diubiquitin, which provides insights into how Rpn13 as a ubiquitin receptor is coupled to substrate deubiquitination by Uch37.

Fragment-Based Drug Discovery of 2-Thiazolidinones as Inhibitors of the Histone Reader BRD4 Bromodomain.

Recognizing acetyllysine of histone is a vital process of epigenetic regulation that is mediated by a protein module called bromodomain. To contribute novel scaffolds for developing into bromodomain inhibitors, we utilize a fragment-based drug discovery approach. By successively applying docking and X-ray crystallography, we were able to identify 9 fragment hits from diffracting more than 60 crystals. In the present work, we described four of them and carried out the integrated lead optimization for fragment 8, which bears a 2-thiazolidinone core. After several rounds of structure guided modifications, we assessed the druggability of 2-thiazolidinone by modulating in vitro pharmacokinetic studies and cellular activity assay. The results showed that two potent compounds of 2-thiazolidinones have good metabolic stability. Also, the cellular assay confirmed the activities of 2-thiazolidinones. Together, we hope the identified 2-thiazolidinone chemotype and other fragment hits described herein can stimulate researchers to develop more diversified bromodomain inhibitors.

Rpn1 provides adjacent receptor sites for substrate binding and deubiquitination by the proteasome.

The yin and yang of proteasomal regulation The ubiquitin-proteasome pathway regulates myriad proteins through their selective proteolysis. The small protein ubiquitin is attached, typically in many copies, to the target protein, which is then recognized and broken down by the proteasome. Shi et al. found a repeat structure in the proteasome for recognizing ubiquitin as well as ubiquitin-like (UBL) proteins. Tandem binding sites allow the proteasome to dock multiple proteins. One of the bound UBL proteins is an enzyme that cleaves ubiquitin-protein conjugates, which antagonizes degradation. Thus, the repetition of related binding sites with distinct specificity achieves a balance of positive and negative regulation of the proteasome. Science, this issue p. 10.1126/science.aad9421 Tandem ligand-binding sites in the proteasome subunit Rpn1 modulate proteasome activity both positively and negatively. INTRODUCTION The ubiquitin-proteasome system comprises hundreds of distinct pathways of degradation, which converge at the step of ubiquitin recognition by the proteasome. Five proteasomal ubiquitin receptors have been identified, two that are intrinsic to the proteasome (Rpn10 and Rpn13) and three reversibly associated proteasomal ubiquitin receptors (Rad23, Dsk2, and Ddi1). RATIONALE We found that the five known proteasomal ubiquitin receptors of yeast are collectively nonessential for ubiquitin recognition by the proteasome. We therefore screened for additional ubiquitin receptors in the proteasome and identified subunit Rpn1 as a candidate. We used nuclear magnetic resonance (NMR) spectroscopy to characterize the structure of the binding site within Rpn1, which we term the T1 site. Mutational analysis of this site showed its functional importance within the context of intact proteasomes. T1 binds both ubiquitin and ubiquitin-like (UBL) proteins, in particular the substrate-delivering shuttle factor Rad23. A second site within the Rpn1 toroid, T2, recognizes the UBL domain of deubiquitinating enzyme Ubp6, as determined by hydrogen-deuterium exchange mass spectrometry analysis and validated by amino acid substitution and functional assays. The Rpn1 toroid thus serves a critical scaffolding role within the proteasome, helping to assemble multiple proteasome cofactors, as well as substrates. RESULTS Our results indicate that proteasome subunit Rpn1 can recognize both ubiquitin and UBL domains of substrate shuttling factors that themselves bind ubiquitin and function as reversibly associated proteasomal ubiquitin receptors. Recognition is mediated by the T1 site within the Rpn1 toroid, which supports proteasome function in vivo. We found that the capacity of T1 to recognize both ubiquitin and UBL shuttling proteins was shared with Rpn10 and Rpn13. The surprising multiplicity of ubiquitin-recognition domains within the proteasome may promote enhanced, multipoint binding of ubiquitin chains. The structures of the T1 site in its free state and in complex with monoubiquitin or lysine 48 (K48)–linked diubiquitin were solved, which revealed that three neighboring outer helices from the T1 toroid engage two ubiquitins. This ubiquitin-binding domain is structurally distinct from those of Rpn10 and Rpn13, despite their common ligands. Moreover, the Rpn1-binding mode leads to a preference for certain ubiquitin chain types, especially K6- and K48-linked chains, in a distinct configuration that can position substrates close to the entry port of the proteasome. The fate of proteasome-docked ubiquitin conjugates is determined by a competition between substrate degradation and deubiquitination; the latter leads to premature release of substrates. Proximal to the T1 site within the Rpn1 toroid is a second UBL-binding site, T2, that does not assist in ubiquitin chain recognition but, rather, in chain disassembly, by binding to the UBL domain of deubiquitinating enzyme Ubp6. Note that the UBL interactors at T1 and T2 are distinct and assign substrate localization to T1 and substrate deubiquitination to T2. CONCLUSION A ligand-binding hotspot was identified in the Rpn1 toroid, consisting of two adjacent receptor sites, referred to as T1 and T2. The Rpn1 toroid represents a distinct class of binding domains for ubiquitin and UBL proteins. The T1 site functions to recruit substrates directly by binding to ubiquitin itself and indirectly by binding to UBL shuttling factors, a feature shared by Rpn10 and Rpn13 despite a lack of structural similarity among these receptors. The T2 site also binds to a UBL domain protein, in this case deubiquitinating enzyme Ubp6. This study thus defines a two-site recognition domain intrinsic to the proteasome that uses distinct ubiquitin-fold ligands to assemble substrates, substrate shuttling factors, and a deubiquitinating enzyme in close proximity. A ligand-binding hotspot in the proteasome for assembling substrates and cofactors. Schematic (top) and model structure (bottom, left) mapping the UBL-binding Rpn1 T1 (indigo) and T2 (orange) sites. (Bottom, right) Enlarged region of the proteasome designed to illustrate Rpn1 T1 and T2 sites bound to a ubiquitinated (yellow) substrate (beige) and deubiquitinating enzyme Ubp6 (green), respectively. Aided by PDB 4CR2, 1WGG, 1VJV, and 2B9R. Hundreds of pathways for degradation converge at ubiquitin recognition by a proteasome. Here, we found that the five known proteasomal ubiquitin receptors in yeast are collectively nonessential for ubiquitin recognition and identified a sixth receptor, Rpn1. A site (T1) in the Rpn1 toroid recognized ubiquitin and ubiquitin-like (UBL) domains of substrate shuttling factors. T1 structures with monoubiquitin or lysine 48 diubiquitin show three neighboring outer helices engaging two ubiquitins. T1 contributes a distinct substrate-binding pathway with preference for lysine 48–linked chains. Proximal to T1 within the Rpn1 toroid is a second UBL-binding site (T2) that assists in ubiquitin chain disassembly, by binding the UBL of deubiquitinating enzyme Ubp6. Thus, a two-site recognition domain intrinsic to the proteasome uses distinct ubiquitin-fold ligands to assemble substrates, shuttling factors, and a deubiquitinating enzyme.

Inhibition of human copper trafficking by a small molecule significantly attenuates cancer cell proliferation.

Copper is a transition metal that plays critical roles in many life processes. Controlling the cellular concentration and trafficking of copper offers a route to disrupt these processes. Here we report small molecules that inhibit the human copper-trafficking proteins Atox1 and CCS, and so provide a selective approach to disrupt cellular copper transport. The knockdown of Atox1 and CCS or their inhibition leads to a significantly reduced proliferation of cancer cells, but not of normal cells, as well as to attenuated tumour growth in mouse models. We show that blocking copper trafficking induces cellular oxidative stress and reduces levels of cellular ATP. The reduced level of ATP results in activation of the AMP-activated protein kinase that leads to reduced lipogenesis. Both effects contribute to the inhibition of cancer cell proliferation. Our results establish copper chaperones as new targets for future developments in anticancer therapies.

Mouse N-acetyltransferase type 2, the homologue of human N-acetyltransferase type 1

There is increasing evidence that human arylamine N-acetyltransferase type 1 (NAT1, EC 2.3.1.5), although first identified as a homologue of a drug-metabolising enzyme, appears to be a marker in human oestrogen receptor positive breast cancer. Mouse Nat2 is the mouse equivalent of human NAT1. The development of mouse models of breast cancer is important, and it is essential to explore the biological role of mouse Nat2. We have therefore produced mouse Nat2 as a recombinant protein and have investigated its substrate specificity profile in comparison with human NAT1. In addition, we have tested the effects of inhibitors on mouse Nat2, including compounds which are endogenous and exogenous steroids. We show that tamoxifen, genistein and diethylstilbestrol inhibit mouse Nat2. The steroid analogue, bisphenol A, also inhibits mouse Nat2 enzymic activity and is shown by NMR spectroscopy, through shifts in proton peaks, to bind close to the active site. A three-dimensional structure for human NAT1 has recently been released, and we have used this crystal structure to generate a model of the mouse Nat2 structure. We propose that a conformational change in the structure is required in order for ligands to bind to the active site of the protein.

Astemizole arrests the proliferation of cancer cells by disrupting the EZH2-EED interaction of polycomb repressive complex 2.

Polycomb Repressive Complex 2 (PRC2) modulates the chromatin structure and transcriptional repression by trimethylation lysine 27 of histone H3 (H3K27me3), a process that necessitates the protein-protein interaction (PPI) between the catalytic subunit EZH2 and EED. Deregulated PRC2 is intimately involved in tumorigenesis and progression, making it an invaluable target for epigenetic cancer therapy. However, until now, there have been no reported small molecule compounds targeting the EZH2-EED interactions. In the present study, we identified astemizole, an FDA-approved drug, as a small molecule inhibitor of the EZH2-EED interaction of PRC2. The disruption of the EZH2-EED interaction by astemizole destabilizes the PRC2 complex and inhibits its methyltransferase activity in cancer cells. Multiple lines of evidence have demonstrated that astemizole arrests the proliferation of PRC2-driven lymphomas primarily by disabling the PRC2 complex. Our findings demonstrate the chemical tractability of the difficult PPI target by a small molecule compound, highlighting the therapeutic promise for PRC2-driven human cancers via targeted destruction of the EZH2-EED complex.

Small-molecule targeting of a diapophytoene desaturase inhibits S. aureus virulence

The surge of antibiotic resistance in Staphylococcus aureus has created a dire need for innovative anti-infective agents that attack new targets, to overcome resistance. In S. aureus, carotenoid pigment is an important virulence factor because it shields the bacterium from host oxidant killing. Here we show that naftifine, a US Food and Drug Administration (FDA)-approved antifungal drug, blocks biosynthesis of carotenoid pigment at nanomolar concentrations. This effect is mediated by competitive inhibition of S. aureus diapophytoene desaturase (CrtN), an essential enzyme for carotenoid pigment synthesis. We found that naftifine attenuated the virulence of a variety of clinical S. aureus isolates, including methicillin-resistant S. aureus (MRSA) strains, in mouse infection models. Specifically, we determined that naftifine is a lead compound for potent CrtN inhibitors. In sum, these findings reveal that naftifine could serve as a chemical probe to manipulate CrtN activity, providing proof of concept that CrtN is a druggable target against S. aureus infections.

Fragment-based drug discovery of 2-thiazolidinones as BRD4 inhibitors: 2. Structure-based optimization.

The signal transduction of acetylated histone can be processed through a recognition module, bromodomain. Several inhibitors targeting BRD4, one of the bromodomain members, are in clinical trials as anticancer drugs. Hereby, we report our efforts on discovery and optimization of a new series of 2-thiazolidinones as BRD4 inhibitors along our previous study. In this work, guided by crystal structure analysis, we reversed the sulfonamide group and identified a new binding mode. A structure-activity relationship study on this new series led to several potent BRD4 inhibitors with IC50 of about 0.05-0.1 μM in FP binding assay and GI50 of 0.1-0.3 μM in cell based assays. To complete the lead-like assessment of this series, we further checked its effects on BRD4 downstream protein c-Myc, investigated its selectivity among five different bromodomain proteins, as well as the metabolic stability test, and reinforced the utility of 2-thiazolidinone scaffold as BET bromodomain inhibitors in novel anticancer drug development.

Metabolomic Analysis of Anti-Hypoxia and Anti-anxiety Effects of Fu Fang Jin Jing Oral Liquid

Background Herba Rhodiolae is a traditional Chinese medicine used by the Tibetan people for treating hypoxia related diseases such as anxiety. Based on the previous work, we developed and patented an anti-anxiety herbal formula Fu Fang Jin Jing Oral Liquid (FJJOL) with Herba Rhodiolae as a chief ingredient. In this study, the anti-hypoxia and anti-anxiety effects of FJJOL in a high altitude forced-swimming mouse model with anxiety symptoms will be elucidated by NMR-based metabolomics. Methods In our experiments, the mice were divided randomly into four groups as flatland group, high altitude saline-treated group, high altitude FJJOL-treated group, and high altitude diazepam-treated group. To cause anxiety effects and hypoxic defects, a combination use of oxygen level decreasing (hypobaric cabin) and oxygen consumption increasing (exhaustive swimming) were applied to mice. After a three-day experimental handling, aqueous metabolites of mouse brain tissues were extracted and then subjected to NMR analysis. The therapeutic effects of FJJOL on the hypobaric hypoxia mice with anxiety symptoms were verified. Results Upon hypoxic exposure, both energy metabolism defects and disorders of functional metabolites in brain tissues of mice were observed. PCA, PLS-DA and OPLS-DA scatter plots revealed a clear group clustering for metabolic profiles in the hypoxia versus normoxia samples. After a three-day treatment with FJJOL, significant rescue effects on energy metabolism were detected, and levels of ATP, fumarate, malate and lactate in brain tissues of hypoxic mice recovered. Meanwhile, FJJOL also up-regulated the neurotransmitter GABA, and the improvement of anxiety symptoms was highly related to this effect. Conclusions FJJOL ameliorated hypobaric hypoxia effects by regulating energy metabolism, choline metabolism, and improving the symptoms of anxiety. The anti-anxiety therapeutic effects of FJJOL were comparable to the conventional anti-anxiety drug diazepam on the hypobaric hypoxia mice. FJJOL might serve as an alternative therapy for the hypoxia and anxiety disorders.