Inhibition of cc chemokine receptor 10 ameliorates osteoarthritis via inhibition of the phosphoinositide-3-kinase/Akt/mammalian target of rapamycin pathway.

BACKGROUND: Osteoarthritis (OA) is a joint disease characterized by inflammation and progressive cartilage degradation. Chondrocyte apoptosis is the most common pathological feature of OA. Interleukin-1ß (IL-1ß), a major inflammatory cytokine that promotes cartilage degradation in OA, often stimulates primary human chondrocytes in vitro to establish an in vitro OA model. Moreover, IL-1ß is involved in OA pathogenesis by stimulating the phosphoinositide-3-kinase (PI3K)/Akt and mitogen-activated protein kinases pathways. The G-protein-coupled receptor, cc chemokine receptor 10 (CCR10), plays a vital role in the occurrence and development of various malignant tumors. However, the mechanism underlying the role of CCR10 in the pathogenesis of OA remains unclear. We aimed to evaluate the protective effect of CCR10 on IL-1ß-stimulated CHON-001 cells and elucidate the underlying mechanism. METHODS: The CHON-001 cells were transfected with a control small interfering RNA (siRNA) or CCR10-siRNA for 24 h, and stimulated with 10 ng/mL IL-1ß for 12 h to construct an OA model in vitro. The levels of CCR10, cleaved-caspase-3, MMP-3, MMP-13, Collagen II, Aggrecan, p-PI3K, PI3K, p-Akt, Akt, phosphorylated-mammalian target of rapamycin (p-mTOR), and mTOR were detected using quantitative reverse transcription polymerase chain reaction and western blotting. Viability, cytotoxicity, and apoptosis of CHON-001 cells were assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, lactate dehydrogenase assay (LDH), and flow cytometry analysis, respectively. Inflammatory cytokines (TNF-α, IL-6, and IL-8) were assessed using enzyme-linked immunosorbent assay. RESULTS: Level of CCR10 was substantially higher in the IL-1ß-stimulated CHON-001 cells than that in the control group, whereas CCR10 was down-regulated in the CCR10-siRNA transfected CHON-001 cells compared to that in the control-siRNA group. Notably, CCR10 inhibition alleviated IL-1ß-induced inflammatory injury in the CHON-001 cells, as verified by enhanced cell viability, inhibited LDH release, reduced apoptotic cells, and cleaved-caspase-3 expression. Meanwhile, IL-1ß induced the release of tumor necrosis factor alpha, IL-6, and IL-8, increase of MMP-3 and MMP-13, and decrease of Collagen II and Aggrecan in the CHON-001 cells, which were reversed by CCR10-siRNA. However, these effects were reversed upon PI3K agonist 740Y-P treatment. Further, IL-1ß-induced PI3K/Akt/mTOR signaling pathway activation was inhibited by CCR10-siRNA, which was increased by 740Y-P treatment. CONCLUSION: Inhibition of CCR10 alleviates IL-1ß-induced chondrocytes injury via PI3K/Akt/mTOR pathway inhibition, suggesting that CCR10 might be a promising target for novel OA therapeutic strategies.

A colorimetric chemosensor for sensitive and selective detection of copper(II) ions based on catalytic oxidation of 1-naphthylamine.

Rapid on-site detection of copper(II) ions (Cu2+) with high sensitivity and selectivity is of great significance in the safety monitoring of drinking water and food. Colorimetric detection is a robust fast determination method with the main drawback of low sensitivity. Herein, we developed a colorimetric chemosensor based on a colored polymer product. Via a Cu-Fenton mechanism, 1-naphthylamine (α-NA) was oxidized by H2O2 and brownish-red poly(1-naphthylamine) (PNA) was produced. The obtained Cu2+ sensor showed a linear response from 0.05 µM to 7 µM, with a detection limit of 62 nM. Our findings expanded chromogenic reaction types for colorimetric detection.

Modulation of plasmonic chiral shell growth on gold nanorods via nonchiral surfactants.

Recently, in combination with seed-mediated growth, thiolated chiral molecule-guided growth has shown great promise in obtaining chiral plasmonic nanostructures. Previously, with the assistance of chiral cysteines (Cys), we realized helical growth of plasmonic shells on gold nanorod (AuNR) seeds dispersed in cetyltrimethylammonium bromide (CTAB) solution. Herein, we further studied the roles of non-chiral cationic surfactants in tuning the helical growth. Both the counter anion and the hydrocarbon chain length of the surfactants were found to affect the formation of helical shells greatly. In particular, we exhibited surfactant-modulated conversion of the chiral shell deposition mode between layer growth and island growth. By optimizing growth conditions, an obvious plasmonic circular dichroism (PCD) response could be achieved for the island helical shell. Our findings demonstrated promising potential of nanochemical synthesis in fabricating chiral plasmonic nanostructures with small structural sizes.

Ancestral body plan and adaptive radiation of sauropterygian marine reptiles.

Sauropterygia is the most diversified and dominant clade among marine reptiles, but their early evolution is scarcely understood. Here we report the earliest known complete specimen related to sauropterygians from the Early Triassic. It is referred to Hanosaurus hupehensis by an exclusive combination of features and shows mosaic morphology integrating the characters of multiple sauropterygian sub-lineages. In phylogenetic results from both parsimony and Bayesian analyses employing our reconstructed dataset, Hanosaurus is stably resolved as the basal-most member of Sauropterygiformes, a clade comprising all sauropterygians and saurosphargids. This skeleton reveals an unexpected ancestral body plan for sauropterygiforms with an elongate trunk and four short limbs, differing from many of its immediate descendants but more similar to non-sauropterygiform marine reptiles at their early aquatic stage as axial swimmers. After this convergence on body plan, we quantitatively confirm the rapid divergence of sauropterygiform reptiles following the end-Permian mass extinction.

Bioinspired Screening of Anti-Adhesion Peptides against Blood Proteins for Intravenous Delivery of Nanomaterials.

Nanomaterials (NMs) inevitably adsorb proteins in blood and form "protein corona" upon intravenous administration as drug carriers, potentially changing the biological properties and intended functions. Inspired by anti-adhesion properties of natural proteins, herein, we employed the one-bead one-compound (OBOC) combinatorial peptide library method to screen anti-adhesion peptides (AAPs) against proteins. The library beads displaying random peptides were screened with three fluorescent-labeled plasma proteins. The nonfluorescence beads, presumed to have anti-adhesion property against the proteins, were isolated for sequence determination. These identified AAPs were coated on gold nanorods (GNRs), enabling significant extension of the blood circulating half-life of these GNRs in mice to 37.8 h, much longer than that (26.6 h) of PEG-coated GNRs. In addition, such AAP coating was found to alter the biodistribution profile of GNRs in mice. The bioinspired screening strategy and resulting peptides show great potential for enhancing the delivery efficiency and targeting ability of NMs.

Progranulin-derived granulin E and lysosome membrane protein CD68 interact to reciprocally regulate their protein homeostasis.

Progranulin (PGRN) is a glycoprotein implicated in several neurodegenerative diseases. It is highly expressed in microglia and macrophages and can be secreted or delivered to the lysosome compartment. PGRN comprises 7.5 granulin repeats and is processed into individual granulin peptides within the lysosome, but the functions of these peptides are largely unknown. Here, we identify CD68, a lysosome membrane protein mainly expressed in hematopoietic cells, as a binding partner of PGRN and PGRN-derived granulin E. Deletion analysis of CD68 showed that this interaction is mediated by the mucin-proline-rich domain of CD68. While CD68 deficiency does not affect the lysosomal localization of PGRN, it results in a specific decrease in the levels of granulin E but no other granulin peptides. On the other hand, the deficiency of PGRN, and its derivative granulin peptides, leads to a significant shift in the molecular weight of CD68, without altering CD68 localization within the cell. Our results support that granulin E and CD68 reciprocally regulate each other's protein homeostasis.

Halofuginone inhibits cell proliferation and AKT/mTORC1 signaling in uterine leiomyoma cells.

The present study aimed to explore the effects of antifibrotic agent halofuginone on uterine leiomyomas (ULs) cells. The survival of the uterine smooth muscle (UtSMC) cells and UL ELT3 cells were measured. Flow cytometry was used to assess the cell cycle distribution and apoptosis. Effects of halofuginone on the state of AKT/mTOR pathway were evaluated. Xenograft animal model was applied to explore the effects of halofuginone in vivo. Halofuginone inhibited the proliferation of ELT3 cells dose-dependently without obvious influence on UtSMC cells. Halofuginone suppressed cell cycle progression and promoted apoptosis of ELT3 cells dose-dependently. Also, p-AKT/AKT and p-p70S6/p70S6 were significantly lowered after treatment with 20 nM halofuginone. Additionally, halofuginone reduced ELT3 tumor growth in xenograft tumor animal model. The present study illustrates that halofuginone inhibits cell proliferation of ULs with low side effects on normal smooth muscle cells, and AKT/mTOR signaling pathway was inactivated meanwhile.

miR-30b-5p inhibits osteoblast differentiation through targeting BCL6.

Human bone marrow mesenchymal stem cells (hBMSCs) are attractive candidates for new therapies to improve bone regeneration and repair. This study was to identify the function of the miR-30b-5p/BCL6 axis in osteogenic differentiation of hBMSCs. Realtime-quantitative PCR (RT-qPCR) and Western blotting were used to measure the relative expression of ALP, OCN, RUNX2, miR-30b-5p, and BCL6 during osteogenic differentiation of hBMSCs. The relationship between miR-30b-5p and BCL6 in hBMSCs was identified using dual-luciferase reporter system and RNA pull-down assay. Alizarin red S staining (ARS) was used to detect the calcium nodules in hBMSCs. We found that the expression of miR-30b-5p was downregulated, whereas that of BCL6 was upregulated during osteogenic differentiation of hBMSCs. Downregulating miR-30b-5p enhanced the expression of OCN, RUNX2, and ALP, and promoted calcium deposition. Conversely, transfection with si-BCL6 had the opposite effect that it inhibited osteogenic differentiation. However, the inhibitory effect of si-BCL6 was abrogated by miR-30b-5p inhibitor. miR-30b-5p inhibits the osteogenic differentiation of hBMSCs by targeting BCL6.

Bottom-Up Synthesis of Helical Plasmonic Nanorods and Their Application in Generating Circularly Polarized Luminescence.

Chiral growth and chirality transfer associated with plasmonic nanostructures have rejuvenated the field of chirality. As the precise regioselective growth of inorganic crystals into chiral shapes at the nanoscale is extremely challenging, "bottom-up" synthesis of intrinsically chiral nanoparticles with structural stability is obviously attractive and important. With the thiolated bimolecular cosurfactants, we demonstrated a chemical strategy for the synthesis of intrinsically helical plasmonic nanorods (HPNRs) with strong and tailorable plasmonic circular dichroism (PCD) responses, deriving from the zwitterionic interactions between the -NH3+ and -COO- groups of the cysteine molecules (Cys). The influence of structural parameters of HPNRs on PCD responses was analyzed systematically by theoretical simulations. Among the different structural parameters, the pitch depth was found to have the greatest impact on the PCD signals, in agreement with the experimental results. Moreover, the obtained HPNRs with the strong, tunable, and stable chiroptical properties were found to be able to induce circularly polarized luminescence of achiral luminophores. Due to the generality of this effect, this chiral plasmonic nanostructure may have great potential for use in the fields of chiral sensors, chiral catalysis, and displays.

Improving peroxidase activity of gold nanorod nanozymes by dragging substrates to the catalysis sites via cysteine modification.

Surface chemistry control is a key means to improve substrate selectivity and enhance catalytic activity of nanozymes, a kind of novel artificial enzymes. Herein, we demonstrated that apart from chemical properties of functional groups, their spatial distance to the catalytic sites is also very important to improve the catalytic performance of nanozymes. Using cetyltrimethylammonium bromide (CTAB) coated gold nanorods (AuNR) as the example, we showed that cysteine (Cys) surface modification can greatly enhance the peroxidase activity of AuNR for the oxidation of substrate 3,3',5,5'-tetramethylbenzidine (TMB). By using cysteine derivatives, the key role of the carboxylic group in cysteine is revealed in improving substrate binding and activity enhancement. The electrostatic interactions of carboxylic groups from adsorbed cysteine molecules with protonated amino groups of TMB bring TMB molecules to the surface Au active sites and thus markedly increase catalytic activity. In contrast, despite having two carboxylic groups, glutathione (GSH) surface modification only leads to quite limited improvement of catalytic activity. We speculated that due to large molecular size of GSH, the spatial distance between TMB-GSH and Au is larger than that between TMB-Cys and Au. Furthermore, Raman characterization indicated that at high Cys coverage, they form patches on rod surface via zwitterionic interactions, which may give additional benefits by decreasing the steric hindrance of TMB diffusion to surface Au atom sites. In all, our study highlights the importance of fine surface tuning in the design of nanozymes.

Analysis of the value of N-terminal pro-B-type natriuretic peptide (NT-proBNP) and other parameters related to right heart function in detecting acute radiation-induced right heart injury.

BACKGROUND: The aim of the present study was to investigate the value of parameters related to right heart function combined with N-terminal pro-B-type natriuretic peptide (NT-proBNP) in acute radiation-induced right heart injury. METHODS: Seventy patients who received chest radiotherapy (RT) in the RT department of our hospital between September 2015 and March 2019 were included in the study. RESULTS: Of the included 70 patients, 19, 32, 4, and 15 had thoracic esophageal cancer, central lung cancer, thymoma, and left breast cancer, respectively. The Tei index, tricuspid annular displacement, right ventricular ejection fraction, and NT-proBNP of the 70 patients were measured 1 week before RT, at weeks 2 and 4 during RT, and 4 weeks after RT. Differences in the Tei index, the tricuspid annular displacement, and NT-proBNP were significant (P<0.01, P<0.05, and P<0.05, respectively). The Tei index significantly increased in the second week of RT. Tricuspid annular displacement decreased significantly 4 weeks after RT. NT-proBNP reached its peak value in the fourth week of RT. However, there was no significant difference in right ventricular ejection fraction (P>0.05). CONCLUSIONS: The Tei index of the right ventricle can be used as a sensitive indicator for the early detection of right heart injury after RT for thoracic tumors. Additionally, tricuspid annular displacement can be used as an index for the early detection of right ventricular damage after RT for thoracic tumors. However, right ventricular ejection fraction showed no significant change in the early stage of right heart damage after RT. Finally, it is important to consider NT-proBNP for the detection of acute radiation-induced heart injury. In acute radiation-induced right heart injury, the combined application of right ventricular Tei index, tricuspid annular displacement, and NT-proBNP is clinically relevant.

Constructing chiral gold nanorod oligomers using a spatially separated sergeants-and-soldiers effect.

A sergeants-and-soldiers (S&S) effect is very useful to the fabrication of supramolecular chirality. This strategy has not yet been explored in the construction of chiral plasmonic superstructures. Herein, we demonstrate a spatially separated S&S effect in fabricating plasmonic superstructures and modulating their chiroptical responses. Specifically, chiral cysteine (Cys) molecules, acting as sergeants, are sandwiched between a gold nanorod (AuNR) core and a Au shell via AuNR-templated Au overgrowth. Cationic surfactants, CTAB (cetyltrimethylammonium bromide) or CPC (cetylpyridinium chloride), are modified on the AuNR@Cys@Au shell surface, thus spatially separating from the chiral sergeants. During the assembly process, the surfactants act as soldiers which could transfer and amplify the local chirality induced by the adsorbed chiral molecules from the plasmonic monomers to the oligomers. Huge PCD signals could be achieved in the plasmonic oligomers by finely tuning chiral sergeants and achiral soldiers, indicating the feasibility of the S&S effect in fabricating chiral plasmonic superstructures.

[Tandem expression of the major epitope domains of the Moschus chrysogaster hemorrhagic disease virus VP60 and its protective efficacy to rabbits].

Moschus chrysogaster (sifanicus) viral hemorrhagic disease (McVHD) is an acute and highly lethal infectious disease caused by Moschus chrysogaster hemorrhagic disease virus (McHDV) whose genome sequence is highly homologous with rabbit hemorrhagic disease virus. To screen the protective antigen of McHDV and set the basis for study of McVHD vaccine, the antigen epitope of major structural protein VP60 of McHDV was analyzed, and the specific primers were designed to obtain three amplified DNA sequences encoding the main antigen epitope of VP60 from McHDV by using RT-PCR. Then the three DNA fragments were sequenced and cloned to prokaryotic expression vector with pET-28a(+) by using overlap extension PCR, and finally the prokaryotic expression plasmid pET-truncated-VP60 was constructed. Subsequently, the pET-truncated-VP60 was transformed into Escherichia coli BL21(DE3), and the recombinant proteins were expressed by IPTG induction. Finally, the expressed protein was purified and applied to immunize that without immunizing with RHD vaccine, then the antiserum titers were evaluated by the hemagglutination inhibition test, and the immune-protective efficacy of the recombinant proteins was observed and analyzed through animal challenge test. The results showed that the multi-epitope DNA fragments of VP60 of McHDV was successfully expressed in the form of inclusion bodies in E. coli, and the relative molecular weight of recombinant proteins is about 45 kDa. After immunized with the recombinant proteins, 100% of New Zealand white rabbits were resistant to attack of McHDV, which indicates efficient immune-protective efficacy of chosen epitope recombinant protein. The study laid a foundation for the development of the new subunit vaccines of McVHD.

Initiation of protective autophagy in hepatocytes by gold nanorod core/silver shell nanostructures.

The high reactivity of silver nanoparticles leads to their broad applications in the anti-bacterial field; however, the safety of silver nanoparticles has attracted increasing public attention. After exposure to silver nanoparticles in vivo, the liver serves as their potential deposition site; however the potential biological effects of such nanoparticles on hepatocytes at low dosages are not well understood. Here, we study the interaction between gold nanorod core/silver shell nanostructures (Au@Ag NRs) and human hepatocytes, HepG2 cells, and determine that Au@Ag NRs at sub-lethal doses can induce autophagy. After uptake, Au@Ag NRs mainly localize in the lysosomes where they release silver ions and promote the production of reactive oxygen species (ROS). The ROS then suppress the AKT-mTOR signaling pathway and activate autophagy. In addition, oxidative stress results in lysosomal impairment, causing decreased ability for lysosomal digestion. Moreover, oxidative stress also affects the structure and function of mitochondria, leading to the initiation of protective autophagy to eliminate the damaged mitochondrion. Our study shows that at sub-lethal dosages, silver nanomaterials may alter the physiological functions of hepatic cells by activating protective autophagy and cause potential health risks, indicating that cautious consideration of the safety of nanomaterials for certain applications is necessary.

Corona of Thorns: The Surface Chemistry-Mediated Protein Corona Perturbs the Recognition and Immune Response of Macrophages.

The significance of protein coronas on the biological fates of nanoparticles has been widely recognized. Therefore, the alterations on biological effects caused by protein coronas need systemic study and interpretation to design novel safe and efficient nanomedicines. In the present study, we present a comprehensive quantitative analysis of the protein coronas on gold nanorods  modified with various surface ligands of different chemical compositions and charges. The design of surface ligands is of utmost importance for the functionalization of nanoparticles, and further, the ligand-induced biological identity determines the fate of nanoparticles in the human body. We found that the surface chemistry influences the composition of the protein corona more profoundly than surface charge. Since the first and most important challenge for administrated nanomedicines is navigating the interaction with macrophages, we further investigated how the surface chemistry-induced specific protein corona affects the phagocytosis and immune responses of macrophages exposed to the corona-nanoparticle complexes. Our results reveal that the protein corona alters the internalization pathways of gold nanorods by macrophages via the interactions of the predominant coronal proteins with specific receptors on the cell membrane. The cytokine secretion profile of macrophages is also highly dependent on the adsorption pattern of the protein corona. The more abundant proteins involved in immune responses, such as acute phase, complement, and tissue leakage proteins, present in the acquired nanoparticle corona, the more macrophage interleukin-1ß (IL-1ß) released is stimulated. The ligand-protein corona composition-immune response coefficient analysis may serve next-generation nanomedicines with high efficiency and good safety for better clinical translation.

Separation of charge carriers and generation of reactive oxygen species by TiO2 nanoparticles mixed with differently-coated gold nanorods under light irradiation.

Combinations of semiconductor nanoparticles (NPs) with noble metal NPs enable an increase in the photoactivity of semiconductor NPs into the visible and near-infrared regions. The design rationale of the semiconductor-metal hybrid nanostructures for the optimization of charge carrier separation and reactive oxygen species (ROS) generation remains unclear. In this study, the interactions of Au nanorods (AuNRs) with TiO2 NPs were modulated by controlling their surface charges. Positively charged AuNRs formed aggregates with the negatively charged TiO2 NPs (AuNR@CTAB/TiO2) upon mixing, suggesting that Schottky junctions may exist between Au and TiO2. In contrast, negatively charged AuNRs (AuNR@PSS) remained spatially separated from the TiO2 NPs in the mixed suspension (AuNR@PSS/TiO2), owing to electrostatic repulsion. We used electron spin resonance (ESR) spectroscopy to detect the separation of charged carriers and ROS generation in these two mixtures under simulated sunlight irradiation. We also explored the role of dissolved oxygen in charge carrier separation and ROS generation by continuously introducing oxygen into the AuNR@CTAB/TiO2 suspension under simulated sunlight irradiation. Moreover, the generation of ROS by the AuNR@CTAB/TiO2 and AuNR@PSS/TiO2 mixtures were also examined under 808 nm laser irradiation. Our results show that the photogenerated electrons of excited semiconductor NPs are readily transferred to noble metal NPs simply by collisions, but the transfer of photogenerated hot electrons from excited AuNRs to TiO2 NPs is more stringent and requires the formation of Schottky junctions. In addition, the introduction of oxygen is an efficient way to enhance the photocatalytic activity of semiconductor NPs/noble metal NPs system combinations.

Cytotoxicity and Cellular Responses of Gold Nanorods to Smooth Muscle Cells Dependent on Surface Chemistry Coupled Action.

Gold nanorods (AuNRs), with their unique physicochemical properties, are recognized as promising materials for biomedical applications. Chemical modification of their surfaces is attracting increasing attention with regard to cytotoxicity and cellular uptake. Herein, the toxicological effects of three types of polymer-coated AuNRs, which are cetyltrimethylammonium bromide-coated AuNRs, polystyrene sulphonate-coated AuNRs, and poly(diallyldimethyl ammonium chloride-coated AuNRs (PDDAC-AuNRs), on vascular smooth muscle cells (VSMCs) are investigated. The results show significantly different effects on VSMCs with different surface coatings. PDDAC-AuNRs, which were nontoxic in cancer cells in previous reports, display extreme toxicity to VSMCs. Initial contact between AuNRs and cell membranes is the important step in AuNRs cellular uptake. Force spectroscopy based on atomic force microscopy is exploited to study interactions between AuNRs and VSMCs membrane in the absence or presence of a corona on the AuNRs surface. The results show that the binding force and binding probability between AuNRs and membranes are closely related to cytotoxicity and cellular responses. These findings highlight the importance of assessing nanoparticle cytotoxicity in somatic cells for medical applications.

Multiscale Analysis of the Microstructure and Stress Evolution in Cold Work Die Steel during Deep Cryogenic Treatment.

Through a combination of 3D representative volume element (RVE) and the metallo-thermo-mechanical coupling finite element (FE) analysis, a multiscale model was established to explore the localized characteristics of microstructure and stress evolution during deep cryogenic treatment (DCT). The results suggest that after cooling to near -160 °C, the largest intensity of martensite is formed, but the retained austenite cannot be eliminated completely until the end of DCT. The driving force for the precipitation of fine and uniform carbides during DCT is provided by the competition between the thermal and phase transformation stresses. Compared with the thermal stress, the phase transformation stress during DCT plays a more significant role. At the interface between retained austenite and martensite, a reduction of around 15.5% retained austenite even induces an obvious increase in the phase transformation stress about 1100 MPa. During DCT, the maximum effective stress in RVE even exceeds 1000 MPa, which may provide a required driving force for the precipitation of fine and homogeneously distributed carbide particles during DCT.

Mechanism of phosphoribosyl-ubiquitination mediated by a single Legionella effector.

Ubiquitination is a post-translational modification that regulates many cellular processes in eukaryotes1-4. The conventional ubiquitination cascade culminates in a covalent linkage between the C terminus of ubiquitin (Ub) and a target protein, usually on a lysine side chain1,5. Recent studies of the Legionella pneumophila SidE family of effector proteins revealed a ubiquitination method in which a phosphoribosyl ubiquitin (PR-Ub) is conjugated to a serine residue on substrates via a phosphodiester bond6-8. Here we present the crystal structure of a fragment of the SidE family member SdeA that retains ubiquitination activity, and determine the mechanism of this unique post-translational modification. The structure reveals that the catalytic module contains two distinct functional units: a phosphodiesterase domain and a mono-ADP-ribosyltransferase domain. Biochemical analysis shows that the mono-ADP-ribosyltransferase domain-mediated conversion of Ub to ADP-ribosylated Ub (ADPR-Ub) and the phosphodiesterase domain-mediated ligation of PR-Ub to substrates are two independent activities of SdeA. Furthermore, we present two crystal structures of a homologous phosphodiesterase domain from the SidE family member SdeD 9 in complexes with Ub and ADPR-Ub. The structures suggest a mechanism for how SdeA processes ADPR-Ub to PR-Ub and AMP, and conjugates PR-Ub to a serine residue in substrates. Our study establishes the molecular mechanism of phosphoribosyl-linked ubiquitination and will enable future studies of this unusual type of ubiquitination in eukaryotes.