Mechanism of nucleosomal H2A K13/15 monoubiquitination and adjacent dual monoubiquitination by RNF168.

The DNA damage repair regulatory protein RNF168, a monomeric RING-type E3 ligase, has a crucial role in regulating cell fate and DNA repair by specific and efficient ubiquitination of the adjacent K13 and K15 (K13/15) sites at the H2A N-terminal tail. However, understanding how RNF168 coordinates with its cognate E2 enzyme UbcH5c to site-specifically ubiquitinate H2A K13/15 has long been hampered by the lack of high-resolution structures of RNF168 and UbcH5c~Ub (ubiquitin) in complex with nucleosomes. Here we developed chemical strategies and determined the cryo-electron microscopy structures of the RNF168-UbcH5c~Ub-nucleosome complex captured in transient H2A K13/15 monoubiquitination and adjacent dual monoubiquitination reactions, providing a 'helix-anchoring' mode for monomeric E3 ligase RNF168 on nucleosome in contrast to the 'compass-binding' mode of dimeric E3 ligases. Our work not only provides structural snapshots of H2A K13/15 site-specific monoubiquitination and adjacent dual monoubiquitination but also offers a near-atomic-resolution structural framework for understanding pathogenic amino acid substitutions and physiological modifications of RNF168.

Sequential release of interacting proteins and Ub-modifying enzymes by disulfide heterotypic ubiquitin reagents.

Heterotypic ubiquitin (Ub) chains have emerged as fundamental components in a wide range of cellular processes. The integrative identification of Ub-interacting proteins (readers) and Ub-modifying enzymes (writers and erasers) that selectively recognize and regulate heterotypic ubiquitination may provide crucial insights into these processes. In this study, we employed the bifunctional molecule-assisted (CAET) strategy to develop a type of disulfide bond-activated heterotypic Ub reagents, which allowed to enrich heterotypic Ub-interacting proteins and modifying enzymes simultaneously. The sequential release of readers which are non-covalently bound and writers or erasers which are covalently conjugated by using urea and reductant, respectively, combined with label-free quantitative (LFQ) MS indicated that these heterotypic Ub reagents would facilitate future investigations into functional roles played by heterotypic Ub chains.

Structure basis for recognition of plant Rpn10 by phytoplasma SAP05 in ubiquitin-independent protein degradation.

Besides traditional ubiquitin-dependent proteasome degradation, thousands of eukaryotic proteins more than previously appreciated could undergo ubiquitin-independent proteasomal degradation (UbInPD). A pathogen-encoded effector protein SAP05 secreted by phytoplasma, could hijack hostage Rpn10 subunit of proteasome derived from Arabidopsis thaliana and target the degradation of GATA BINDING FACTOR (GATA) or SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors (TFs) without ubiquitin or additional proteasome shuttle factors. To explain how could SAP05 target the degradation bypassing the ubiquitin-dependent pathway, we have determined the crystal structure of the complex between Arabidopsis thaliana Rpn10 and Aster Yellows witches'-broom phytoplasma SAP05 or onion yellow phytoplasma SAP05, which showed a previously unknown recognition interface. Sequence alignment and structural biological evidence showed that this interaction is highly conserved in various SAP05 homologs, suggesting a general mode in plant infection. After docking the complex structure to the plant proteasome, SAP05 was near to the adenosine triphosphatase (ATPase) central pore and enough to submit substrate to degradation process, which suggested a molecular glue-like role to bridge TFs close to the ATPase central pore of proteasomes for the direct degradation.

PC 18:1/18:1 mediates the anti-inflammatory effects of exercise and remodels tumor microenvironment of hepatocellular carcinoma.

AIM: Phosphatidylcholine (PC) is essential for membrane structural integrity and lipid-dependent signaling pathways, and is an essential component required for cancer cell growth. Using hepatocellular carcinoma (HCC) as a tumor model, this study aims to further screen phospholipid biomarkers of the tumor microenvironment and explore the anti-tumor effects and mechanisms of aerobic exercise. MAIN METHODS: The HCC of C57BL/6J mice was induced by the injection of the carcinogen diethylnitrosamine (DEN). Exercise was performed on an ungraded treadmill for weeks. The inflammation-related markers were detected by ELISA, PCR and immunohistochemistry, hepatic metabolic profile was analyzed by GC/MS, and lipid metabolism profile was further detected by lipid-targeted LC/MS. Cell culture was used to verify the anti-inflammatory effect of PC. KEY FINDINGS: Exercise reduced hepatic inflammation, tumor incidence and volume. Metabolomics analysis showed that palmitic acid is a key metabolic marker for exercise to improve tumor microenvironment. Injection of exogenous palmitic acid following exercise impaired the anti-inflammatory and anti-tumor effects of exercise. Lipid metabolomics analysis further showed that metabolites for exercise were enriched in glycerol phospholipid metabolism, including 14 phosphatidylcholines (PCs), 18 phosphatidylethanolamines (PEs), and 6 triglycerides (TGs). These biomarkers contain different lengths of fatty acid chains and different numbers of unsaturated bonds, respectively. Cell culture verified that PC (18:1/18:1) mediated lipopolysaccharide (LPS)-induced inflammation in HepG2 cell. SIGNIFICANCE: Our results suggest that exercise remodels glycerophospholipid metabolism and reduces hepatic palmitic acid loading and PC (18:1/18:1) level, thereby reconstructing a microenvironment that is hostile to HCC.

Improved gene therapy for MFRP deficiency-mediated retinal degeneration by knocking down endogenous bicistronic Mfrp and Ctrp5 transcript.

The membrane frizzled-related protein (Mfrp) and C1-tumor necrosis factor related protein 5 (Ctrp5) genes are transcribed as a bicistronic unit and dysregulation of either gene is associated with retinal degeneration in the retinal pigment epithelium (RPE) cells. However, the mechanisms that regulate the expression of the bicistronic transcript remain controversial. Here, we identified a microRNA-based negative feedback loop that helps maintain a normal expression level of the bicistronic Mfrp and Ctrp5 transcript. Specifically, miR-149-3p, a conserved microRNA, binds to the 3'UTR of the Mfrp gene. In MFRP-deficient rd6 mice, the miR-149-3p levels were compromised compared with those in WT mice, resulting in an increase in the bicistronic transcript. We also report a capsid-modified rAAVDJ-3M vector that is capable of robustly and specifically transducing RPE cells following subretinal delivery. Compared with the parental vector, the modified vector elicited similar levels of serum anti-rAAV antibodies, but recruited fewer microglial infiltrations. Most significantly, we also demonstrate that simultaneous overexpressing of MFRP and knockdown of the bicistronic transcript was more effective in rescuing vision than MFRP overexpression alone. Our findings offer new insights into the function of MFRP and provide a promising therapeutic strategy for the treatment of MFRP-associated ocular diseases.

Spatial Correlation Network and Driving Factors of Urban Energy Eco-Efficiency from the Perspective of Human Well-Being: A Case Study of Shaanxi Province, China.

It is very important to seek a sustainable improvement in human well-being under a limited resource supply and to promote the scientific and coordinated development of urban economic development, ecological environment protection, and human well-being. This paper constructs a human well-being index that includes economic well-being, culture and education well-being, and social development well-being as factors, and it incorporates the human well-being index into the evaluation system for urban well-being energy eco-efficiency (WEE). It uses the super-slack-based measure (SBM) model, which considers undesirable output, to measure the WEE of 10 prefecture-level cities in Shaanxi Province, China, from 2005 to 2019. The social network analysis (SNA) is used to describe the characteristics of the spatial correlation network of WEE and its spatiotemporal evolutionary trend, and the quadratic assignment procedure (QAP) analysis method is used to identify the driving factors that affect the spatial correlation network. The results show that, first, the WEE in Shaanxi is relatively low as a whole and varies greatly among regions, with the highest level in northern Shaanxi, followed by Guanzhong; the lowest level is in southern Shaanxi. Second, in Shaanxi, WEE has transcended geographical proximity into a complex, multi-threaded spatial correlation network, and Yulin is at the center of the network. Third, the network shows four sectors: the net overflow, main benefit, two-way overflow, and broker. Members in each sector have not fully exploited their advantages, and the whole network can be improved. Fourth, the differences in the economic development level, openness, industrial structure, and population are the main driving factors influencing the formation of the spatial correlation network.

A bifunctional molecule-assisted synthesis of mimics for use in probing the ubiquitination system.

Ubiquitination regulates almost every life process of eukaryotes. The study of the ubiquitin (Ub) coupling or decoupling process and the interaction study of Ub-Ub binding protein have always been the central focus. However, such studies are challenging, owing to the transient nature of Ub-coupling enzymes and deubiquitinases in the reactions, as well as the difficulty in preparing large quantities of polyubiquitinated samples. Here we describe a recently developed strategy for the efficient preparation of analogs of Ub chains and analogs for Ub coupling and uncoupling intermediates, which facilitate the study of the ubiquitination process. The strategy includes mainly the following steps: (i) the bifunctional molecule conjugation on the only cysteine (Cys) residue of a target protein (usually a Ub or Ub-conjugating enzyme), exposing an orthogonal reactive site for native chemical ligation; (ii) covalent ligation with a Ub-derived thioester, exposing a free sulfhydryl; and (iii) (optional) a disulfide bond formation with a substrate protein (mainly with only one Cys protein) through nonactivity-based cross-linking or with a deubiquitinase (mainly with several Cys residues) through activity-based cross-linking. When the bifunctional molecule and target proteins are obtained, the final products can be prepared in milligram quantities within 2 weeks.

The use of melittin to enhance transgene expression mediated by recombinant adeno-associated virus serotype 2 vectors both in vitro and in vivo.

OBJECTIVE: Melittin, a cell-penetrating peptide, improves the efficiency of many non-viral gene delivery vectors, yet its application in viral vectors has not been well studied. The non-pathogenic recombinant adeno-associated virus (rAAV) vector is an ideal in vivo gene delivery vector. However, its full potential will only be achieved after improvement of its transduction efficiency. To improve the transduction efficiency of rAAV2 vectors, we attempted to develop a melittin-based rAAV2 vector delivery strategy. METHODS: The melittin peptide was inserted into the rAAV2 capsid either in the loop VIII of all viral proteins (VPs) or at the N terminus of VP2. Various rAAV2-gfp or -fluc vectors were subjected to quantitative real-time polymerase chain reaction and Western blot assays to determine their titers and integrity of capsid proteins, respectively. Alternatively, the vectors based on wild-type capsid were pre-incubated with melittin, followed by transduction of cultured cells or tail vein administration of the mixture to C57BL/6 and BALB/c nude mice. In vivo bioluminescence imaging was performed to evaluate the transgene expression. RESULTS: rAAV2 vectors with melittin peptide inserted in the loop VIII of VPs had low transduction efficiency, probably due to dramatically reduced ability to bind to the target cells. Fusing the melittin peptide at the N-terminus of VP2 produced vectors without the VP2 subunit. Interestingly, among the commonly used rAAV vectors, pre-incubation of rAAV2 and rAAV6 vectors with melittin significantly enhanced their transduction efficiency in HEK293 and Huh7 cells in vitro. Melittin also had the ability to increase the rAAV2-mediated transgene expression in mouse liver in vivo. Mechanistically, melittin did not change the vector-receptor interaction. Moreover, cell counting kit-8 assays of cultured cells and serum transaminase levels indicated melittin had little cytotoxicity. CONCLUSION: Pre-incubation with melittin, but not insertion of melittin into the rAAV2 capsid, significantly enhanced rAAV2-mediated transgene expression. Although further in vivo evaluations are required, this research not only expands the pharmacological potential of melittin, but also provides a new strategy to improve gene therapy mediated by rAAV vectors.

Expedient Synthesis of Ubiquitin-like Protein ISG15 Tools through Chemo-Enzymatic Ligation Catalyzed by a Viral Protease Lbpro.

Ubiquitin (Ub)-like protein ISG15 (interferon-stimulated gene 15) regulates innate immunity and links with the evasion of host response by viruses such as SARS-CoV-2. Dissecting ISGylation pathways recently received increasing attention which can inform related disease interventions, but such studies necessitate the preparation and development of various ISG15 protein tools. Here, we find that the leader protease (Lbpro ) encoded by foot-and-mouth disease virus can promote ligation reactions between recombinant ISG15 and synthetic glycyl compounds, generating protein tools such as ISG15-propargylamide and ISG15-rhodamine110, which are needed for cellular proteomic studies of deISGylases, and the screening and evaluation of inhibitors against SARS-CoV-2 papain-like protease (PLpro). Furthermore, this strategy can be also used to load ISG15 onto the lysine of a synthetic peptide through an isopeptide bond, and prepare Ub and NEDD8 (ubiquitin-like protein Nedd8) protein tools.

Recent progress in dissecting ubiquitin signals with chemical biology tools.

Protein ubiquitination regulates almost all eukaryotic cellular processes, and is of very high complexity due to the diversity of ubiquitin (Ub) modifications including mono-, multiply mono-, homotypic poly-, and even heterotypic poly-ubiquitination. To accurately elucidate the role of each specific Ub signal in different cells with spatiotemporal resolutions, a variety of chemical biology tools have been developed. In this review, we summarize some recently developed chemical biology tools for ubiquitination studies and their applications in molecular and cellular biology.

Associations of Gut Microbiota and Fatty Metabolism With Immune Thrombocytopenia.

Objective: To determine whether gut microbiota, fatty metabolism and cytokines were associated with immune thrombocytopenia (ITP). Methods: In total, 29 preliminarily diagnosed ITP patients and 33 healthy volunteers were enrolled. Fecal bacterial were analyzed based on 16S rRNA sequencing. Plasma cytokines and motabolites were analyzed using flow cytometry and liquid chromatography-mass spectrometry (LC-MS), respectively. Results: Bacteroides, Phascolarctobacterium, and Lactobacillus were enriched at the genus level in ITP patients, while Ruminococcaceae UCG-002, Eubacterium coprostanoligeues, Megamonas, and Lachnospiraceae NC2004 were depleted. At the phylum level, the relative abundance of Proteobacteria and Chloroflexi increased in ITP patients, while Firmicutes, Actinobacteria, and the Firmicutes/Bacteroidetes ratio decreased. Plasma levels of 5-hydroxyeicosatetraenoic acid (5-HETE), 6-trans-12-epi-leukotriene B4 (6t,12e-LTB4), and resolvin D2 (RvD2) were upregulated, and stachydrine, dowicide A, dodecanoylcarnitine were downregulated in ITP patients. Furthermore, RvD2 is positively correlated with order Bacteroidetes VC2.1 Bac22, 5-HETE is positively correlated with genus Azospirillum, and 6t,12e-LTB4 is positively correlated with genus Cupriavidus. In addition, stachydrine is positively correlated with family Planococcaceae, dowicide A is positively correlated with class MVP-15, and dodecanoylcarnitine is positively correlated with order WCHB1-41. Plasma levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were upregulated in ITP patients. Conclusion: Our study revealed a relationship between microbiota and fatty metabolism in ITP. Gut microbiota may participate in the pathogenesis of ITP through affecting cytokine secretion, interfering with fatty metabolism.

Thioester-Assisted Sortase-A-Mediated Ligation.

Sortase A (SrtA)-mediated ligation, a popular method for protein labeling and semi-synthesis, is limited by its reversibility and dependence on the LPxTG motif, where "x" is any amino acid. Here, we report that SrtA can mediate the efficient and irreversible ligation of a protein/peptide containing a C-terminal thioester with another protein/peptide bearing an N-terminal Gly, with broad tolerance for a wide variety of LPxT-derived sequences. This strategy, the thioester-assisted SrtA-mediated ligation, enabled the expedient preparation of proteins bearing various N- or C-terminal labels, including post-translationally modified proteins such as the Ser139-phosphorylated histone H2AX and Lys9-methylated histone H3, with less dependence on the LPxTG motif. Our study validates the chemical modification of substrates as an effective means of augmenting the synthetic capability of existing enzymatic methods.

Anabolic Bone Stimulus Requires a Pre-Exercise Meal and 45-Minute Walking Impulse of Suprathreshold Speed-Enhanced Momentum to Prevent or Mitigate Postmenopausal Osteoporosis within Circadian Constraints.

Osteoporosis currently afflicts 8 million postmenopausal women in the US, increasing the risk of bone fractures and morbidity, and reducing overall quality of life. We sought to define moderate exercise protocols that can prevent postmenopausal osteoporosis. Our previous findings singled out higher walking speed and pre-exercise meals as necessary for suppression of bone resorption and increasing of markers of bone formation. Since both studies were amenable to alternate biomechanical, nutritional, and circadian interpretations, we sought to determine the relative importance of higher speed, momentum, speed-enhanced load, duration of impulse, and meal timing on osteogenic response. We hypothesized that: (1) 20 min of exercise one hour after eating is sufficient to suppress bone resorption as much as a 40-min impulse and that two 20 min exercise bouts separated by 7 h would double the anabolic effect; (2) early morning exercise performed after eating will be as effective as mid-day exercise for anabolic outcome; and (3) the 08:00 h 40-min. exercise uphill would be as osteogenic as the 40-min exercise downhill. Healthy postmenopausal women, 8 each, were assigned to a no-exercise condition (SED) or to 40- or 20-min exercise bouts, spaced 7 h apart, for walking uphill (40 Up and 20 Up) or downhill (40 Down and 20 Down) to produce differences in biomechanical variables. Exercise was initiated at 08:00 h one hour after eating in 40-min groups, and also 7 h later, two hours after the midday meal, in 20-min groups. Measurements were made of CICP (c-terminal peptide of type I collagen), osteocalcin (OC), and bone-specific alkaline phosphatase (BALP), markers of bone formation, and of the bone resorptive marker CTX (c-terminal telopeptide of type 1 collagen). The osteogenic ratios CICP/CTX, OC/CTX, and BALP/CTX were calculated. Only the 40-min downhill exercise of suprathreshold speed-enhanced momentum, increased the three osteogenic ratios, demonstrating the necessity of a 40-min, and inadequacy of a 20-min, exercise impulse. The failure of anabolic outcome in 40-min uphill exercise was attributed to a sustained elevation of PTH concentration, as its high morning elevation enhances the CTX circadian rhythm. We conclude that postmenopausal osteoporosis can be prevented or mitigated in sedentary women by 45 min of morning exercise of suprathreshold speed-enhanced increased momentum performed shortly after a meal while walking on level ground, or by 40-min downhill, but not 40-min uphill, exercise to avoid circadian PTH oversecretion. The principal stimulus for the anabolic effect is exercise, but the prerequisite for a pre-exercise meal demonstrates the requirement for nutrient facilitation.

Structural insights into Ubr1-mediated N-degron polyubiquitination.

The N-degron pathway targets proteins that bear a destabilizing residue at the N terminus for proteasome-dependent degradation1. In yeast, Ubr1-a single-subunit E3 ligase-is responsible for the Arg/N-degron pathway2. How Ubr1 mediates the initiation of ubiquitination and the elongation of the ubiquitin chain in a linkage-specific manner through a single E2 ubiquitin-conjugating enzyme (Ubc2) remains unknown. Here we developed chemical strategies to mimic the reaction intermediates of the first and second ubiquitin transfer steps, and determined the cryo-electron microscopy structures of Ubr1 in complex with Ubc2, ubiquitin and two N-degron peptides, representing the initiation and elongation steps of ubiquitination. Key structural elements, including a Ubc2-binding region and an acceptor ubiquitin-binding loop on Ubr1, were identified and characterized. These structures provide mechanistic insights into the initiation and elongation of ubiquitination catalysed by Ubr1.

The use of miR122 and its target sequence in adeno-associated virus-mediated trichosanthin gene therapy.

OBJECTIVE: Plant-derived cytotoxic transgene expression, such as trichosanthin (tcs), regulated by recombinant adeno-associated virus (rAAV) vector is a promising cancer gene therapy. However, the cytotoxic transgene can hamper the vector production in the rAAV producer cell line, human embryonic kidney (HEK293) cells. Here, we explored microRNA-122 (miR122) and its target sequence to limit the expression of the cytotoxic gene in the rAAV producer cells. METHODS: A miR122 target (122T) sequence was incorporated into the 3' untranslated region of the tcs cDNA sequence. The firefly luciferase (fluc) transgene was used as an appropriate control. Cell line HEK293-mir122 was generated by the lentiviral vector-mediated genome integration of the mir122 gene in parental HEK293 cells. The effects of miR122 overexpression on cell growth, transgene expression, and rAAV production were determined. RESULTS: The presence of 122T sequence significantly reduced transgene expression in the miR122-enriched Huh7 cell line (in vitro), fresh human hepatocytes (ex vivo), and mouse liver (in vivo). Also, the normal liver physiology was unaffected by delivery of 122T sequence by rAAV vectors. Compared with the parental cells, the miR122-overexpressing HEK293-mir122 cell line showed similar cell growth rate and expression of transgene without 122T, as well as the ability to produce liver-targeting rAAV vectors. Fascinatingly, the yield of rAAV vectors carrying the tcs-122T gene was increased by 77.7-fold in HEK293-mir122 cells. Moreover, the tcs-122T-containing rAAV vectors significantly reduced the proliferation of hepatocellular carcinoma cells without affecting the normal liver cells. CONCLUSION: HEK293-mir122 cells along with the 122T sequence provide a potential tool to attenuate the cytotoxic transgene expression, such as tcs, during rAAV vector production.

Low endotoxin E. coli strain-derived plasmids reduce rAAV vector-mediated immune responses both in vitro and in vivo.

The major challenge of recombinant adeno-associated virus (rAAV) vectors is host immunological barriers. Compared to the neutralizing antibody and the cytotoxic T lymphocyte response, the host immune responses induced by unsatisfactory rAAV manufacturing were largely ignored previously. rAAV vector production usually requires large amounts of plasmid DNAs. The DNA are commonly isolated from the DH5α bacterial strain, which contains lipopolysaccharide (LPS) contamination. LPS, also named endotoxin, in plasmid DNA is intractable, and residual endotoxin in the subsequent rAAV vectors may result in substantial host immune response. Recently, a ClearColi K12 bacterial strain is commercially available, with genetically modified LPS that does not trigger endotoxic response in mammalian cells. Here, we produced rAAV-DJ vectors by plasmids yielded from either DH5α or ClearColi K12 bacterial strains. Our data indicated that the ClearColi K12 strain had satisfactory protection for the rAAV inverted terminal repeat (ITR) sequence. As expected, the ClearColi K12-derived rAAV-DJ vectors had lower endotoxin levels. The physical and biological equivalency of the purified viral stocks were confirmed by electron micrographs, Coomassie blue staining, and transduction assays. Most importantly, the ClearColi K12-derived rAAV-DJ vectors triggered reduced nuclear factor-kappa B (NF-κB) signaling pathway both in cell cultures in vitro and in C57BL/6 mice retinas in vivo. We believe that the use of the ClearColi K12 bacterial strain could eliminate the LPS in the purified vector stock at the source. Our data indicate its promising use in future clinical development.

K29-linked ubiquitin signaling regulates proteotoxic stress response and cell cycle.

Protein ubiquitination shows remarkable topological and functional diversity through the polymerization of ubiquitin via different linkages. Deciphering the cellular ubiquitin code is of central importance to understand the physiology of the cell. However, our understanding of its function is rather limited due to the lack of specific binders as tools to detect K29-linked polyubiquitin. In this study, we screened and characterized a synthetic antigen-binding fragment, termed sAB-K29, that can specifically recognize K29-linked polyubiquitin using chemically synthesized K29-linked diubiquitin. We further determined the crystal structure of this fragment bound to the K29-linked diubiquitin, which revealed the molecular basis of specificity. Using sAB-K29 as a tool, we uncovered that K29-linked ubiquitination is involved in different kinds of cellular proteotoxic stress response as well as cell cycle regulation. In particular, we showed that K29-linked ubiquitination is enriched in the midbody and downregulation of the K29-linked ubiquitination signal arrests cells in G1/S phase.

Mechanistic insight into substrate processing and allosteric inhibition of human p97.

p97 processes ubiquitinated substrates and plays a central role in cellular protein homeostasis. Here, we report a series of cryo-EM structures of the substrate-engaged human p97 complex with resolutions ranging from 2.9 to 3.8 Å that captured 'power-stroke'-like motions of both the D1 and D2 ATPase rings of p97. A key feature of these structures is the critical conformational changes of the intersubunit signaling (ISS) motifs, which tighten the binding of nucleotides and neighboring subunits and contribute to the spiral staircase conformation of the D1 and D2 rings. In addition, we determined the cryo-EM structure of human p97 in complex with NMS-873, a potent p97 inhibitor, at a resolution of 2.4 Å. The structures showed that NMS-873 binds at a cryptic groove in the D2 domain and interacts with the ISS motif, preventing its conformational change and thus blocking substrate translocation allosterically.

Chemical Synthesis of Activity-Based E2-Ubiquitin Probes for the Structural Analysis of E3 Ligase-Catalyzed Transthiolation.

Activity-based E2 conjugating enzyme (E2)-ubiquitin (Ub) probes have recently emerged as effective tools for studying the molecular mechanism of E3 ligase (E3)-catalyzed ubiquitination. However, the preparation of existing activity-based E2-Ub probes depends on recombination technology and bioconjugation chemistry, limiting their structural diversity. Herein we describe an expedient total chemical synthesis of an E2 enzyme variant through a hydrazide-based native chemical ligation, which enabled the construction of a structurally new activity-based E2-Ub probe to covalently capture the catalytic site of Cys-dependent E3s. Chemical cross-linking coupled with mass spectrometry (CXMS) demonstrated the utility of this new probe in structural analysis of the intermediates formed during Nedd4 and Parkin-mediated transthiolation. This study exemplifies the utility of chemical protein synthesis for the development of protein probes for biological studies.

Seesaw conformations of Npl4 in the human p97 complex and the inhibitory mechanism of a disulfiram derivative.

p97, also known as valosin-containing protein (VCP) or Cdc48, plays a central role in cellular protein homeostasis. Human p97 mutations are associated with several neurodegenerative diseases. Targeting p97 and its cofactors is a strategy for cancer drug development. Despite significant structural insights into the fungal homolog Cdc48, little is known about how human p97 interacts with its cofactors. Recently, the anti-alcohol abuse drug disulfiram was found to target cancer through Npl4, a cofactor of p97, but the molecular mechanism remains elusive. Here, using single-particle cryo-electron microscopy (cryo-EM), we uncovered three Npl4 conformational states in complex with human p97 before ATP hydrolysis. The motion of Npl4 results from its zinc finger motifs interacting with the N domain of p97, which is essential for the unfolding activity of p97. In vitro and cell-based assays showed that the disulfiram derivative bis-(diethyldithiocarbamate)-copper (CuET) can bypass the copper transporter system and inhibit the function of p97 in the cytoplasm by releasing cupric ions under oxidative conditions, which disrupt the zinc finger motifs of Npl4, locking the essential conformational switch of the complex.