Metabolic Regulation of Gene Expression by Histone Lysine ß-Hydroxybutyrylation.

Here we report the identification and verification of a ß-hydroxybutyrate-derived protein modification, lysine ß-hydroxybutyrylation (Kbhb), as a new type of histone mark. Histone Kbhb marks are dramatically induced in response to elevated ß-hydroxybutyrate levels in cultured cells and in livers from mice subjected to prolonged fasting or streptozotocin-induced diabetic ketoacidosis. In total, we identified 44 histone Kbhb sites, a figure comparable to the known number of histone acetylation sites. By ChIP-seq and RNA-seq analysis, we demonstrate that histone Kbhb is a mark enriched in active gene promoters and that the increased H3K9bhb levels that occur during starvation are associated with genes upregulated in starvation-responsive metabolic pathways. Histone ß-hydroxybutyrylation thus represents a new epigenetic regulatory mark that couples metabolism to gene expression, offering a new avenue to study chromatin regulation and diverse functions of ß-hydroxybutyrate in the context of important human pathophysiological states, including diabetes, epilepsy, and neoplasia.

SNP-adjacent super enhancer network mediates enhanced osteogenic differentiation of MSCs in ankylosing spondylitis.

Ankylosing spondylitis (AS) is a rheumatic disease with pathological osteogenesis that causes bony ankylosis and even deformity over time. Mesenchymal stem cells (MSCs) are multipotent stem cells that are the main source of osteoblasts. We previously demonstrated that enhanced osteogenic differentiation of MSCs from AS patients (ASMSCs) is related to pathological osteogenesis in AS. However, the more concrete mechanism needs further exploration. Super enhancers (SEs) are dense clusters of stitched enhancers that control cell identity determination and disease development. Single-nucleotide polymorphisms (SNPs) regulate the formation and interaction of SEs and denote genes accounting for AS susceptibility. Via integrative analysis of multiomic data, including histone 3 lysine 27 acetylation (H3K27ac), chromatin immunoprecipitation sequencing (ChIP-seq), SNPs and RNA sequencing (RNA-seq) data, we discovered a transcription network mediated by AS SNP-adjacent SEs (SASEs) in ASMSCs and identified key genes, such as Toll-like receptor 4 (TLR4), interleukin 18 receptor 1 (IL18R1), insulin-like growth factor binding protein 4 (IGFBP4), transportin 1 (TNPO1) and proprotein convertase subtilisin/kexin type 5 (PCSK5), which are pivotal in osteogenesis and AS pathogenesis. The SASE-regulated network modulates the enhanced osteogenic differentiation of ASMSCs by synergistically activating the PI3K-Akt, NF-kappaB and Hippo signaling pathways. Our results emphasize the crucial role of the SASE-regulated network in pathological osteogenesis in AS, and the preferential inhibition of ASMSC osteogenic differentiation by JQ1 indicates that SEs may be attractive targets in future treatment for new bone formation in AS.

DAPK1 Interacts with the p38 Isoform MAPK14, Preventing Its Nuclear Translocation and Stimulation of Bone Marrow Adipogenesis.

Bone marrow (BM) adipose tissue (BMAT), a unique adipose depot, plays an important role in diseases such as osteoporosis and bone metastasis. Precise control of mesenchymal stem cell (MSC) differentiation is critical for BMAT formation and regeneration. Here, we show that death associated protein kinase 1 (DAPK1) negatively regulates BM adipogenesis in vitro and in vivo. Prx1creDapk1loxp/loxp mice showed more adipocytes in the femur than Dapk1loxp/loxp mice. Further mechanistic analyses revealed that DAPK1 inhibits p38 mitogen-activated protein kinase (MAPK) signaling in the nucleus by binding the p38 isoform MAPK14, decreasing p38 nuclear activity, which subsequently inhibits BM adipogenesis. The inhibitory effect of DAPK1 against MAPK14 was independent of its kinase activity. In addition, the decreased DAPK1 was observed in the BM-MSCs of ageing mice. Our results reveal a previously undescribed function for DAPK1 in the regulation of adipogenesis and may also reveal the underlying mechanism of BMAT formation in ageing.

A novel Anti-ROS osteoblast-specific delivery system for ankylosing spondylitis treatment via suppression of both inflammation and pathological new bone formation.

Ankylosing spondylitis (AS) is a common rheumatic disorder distinguished by chronic inflammation and heterotopic ossification at local entheses sites. Currently available medications, including nonsteroidal anti-inflammatory drugs (NSAIDs), disease-modifying anti-rheumatic drugs (DMARDs) and TNF inhibitors, are limited by side effects, high costs and unclear inhibitory effects on heterotopic ossification. Herein, we developed manganese ferrite nanoparticles modified by the aptamer CH6 (CH6-MF NPs) that can efficiently scavenge ROS and actively deliver siRNA into hMSCs and osteoblasts in vivo for effective AS treatment. CH6-MF NPs loaded with BMP2 siRNA (CH6-MF-Si NPs) effectively suppressed abnormal osteogenic differentiation under inflammatory conditions in vitro. During their circulation and passive accumulation in inflamed joints in the Zap70mut mouse model, CH6-MF-Si NPs attenuated local inflammation and rescued heterotopic ossification in the entheses. Thus, CH6-MF NPs may be an effective inflammation reliever and osteoblast-specific delivery system, and CH6-MF-Si NPs have potential for the dual treatment of chronic inflammation and heterotopic ossification in AS.

Targeting macrophage M1 polarization suppression through PCAF inhibition alleviates autoimmune arthritis via synergistic NF-κB and H3K9Ac blockade.

Sustained inflammatory invasion leads to joint damage and progressive disability in several autoimmune rheumatic diseases. In recent decades, targeting M1 macrophage polarization has been suggested as a promising therapeutic strategy for autoimmune arthritis. P300/CBP-associated factor (PCAF) is a histone acetyltransferase (HAT) that exhibits a strong positive relationship with the proinflammatory microenvironment. However, whether PCAF mediates M1 macrophage polarization remains poorly studied, and whether targeting PCAF can protect against autoimmune arthritis in vivo remains unclear. Commonly used drugs can cause serious side effects in patients because of their extensive and nonspecific distribution in the human body. One strategy for overcoming this challenge is to develop drug nanocarriers that target the drug to desirable regions and reduce the fraction of drug that reaches undesirable targets. In this study, we demonstrated that PCAF inhibition could effectively inhibit M1 polarization and alleviate arthritis in mice with collagen-induced arthritis (CIA) via synergistic NF-κB and H3K9Ac blockade. We further designed dextran sulfate (DS)-based nanoparticles (DSNPs) carrying garcinol (a PCAF inhibitor) to specifically target M1 macrophages in inflamed joints of the CIA mouse model via SR-A-SR-A ligand interactions. Compared to free garcinol, garcinol-loaded DSNPs selectively targeted M1 macrophages in inflamed joints and significantly improved therapeutic efficacy in vivo. In summary, our study indicates that targeted PCAF inhibition with nanoparticles might be a promising strategy for treating autoimmune arthritis via M1 macrophage polarization inhibition.

Galangin mitigates glucocorticoid-induced osteoporosis by activating autophagy of BMSCs via triggering the PKA/CREB signaling pathway.

Glucocorticoid-induced osteoporosis (GIOP), one of the most common and serious adverse effects associated with glucocorticoid administration, manifests as decreased bone formation and increased bone resorption, eventually culminating in bone loss. Galangin (GAL) is a flavonoid extracted from the medicinal herbal galangal that possesses a variety of pharmacological activities and can inhibit osteoclastogenesis. However, the effects of GAL on GIOP remain unclear. Our study aims to explore the effects of GAL on GIOP in mice and the underlying mechanism. Our results show that GAL markedly mitigates the severity of dexamethasone (Dex)-induced osteoporosis in mice and potentiates osteogenic differentiation in mouse bone marrow-derived mesenchymal stem cells (BMSCs). Furthermore, GAL also significantly counteracts Dex-mediated suppression of osteogenic differentiation and autophagy in human BMSCs. GAL augments PKA/CREB-mediated autophagic flux in BMSCs and the bones of osteoporotic mice. GAL-mediated osteogenic differentiation in Dex-treated BMSCs is significantly decreased by the PKA inhibitor H89 and autophagy inhibitor 3-methyladenine. Collectively, our data indicate that GAL can ameliorate GIOP, partly by augmenting the mineralization of BMSCs by potentiating PKA/CREB-mediated autophagic flux, highlighting its potential therapeutic use in treating glucocorticoid-related osteoporosis.

Alpinetin alleviates osteoporosis by promoting osteogenic differentiation in BMSCs by triggering autophagy via PKA/mTOR/ULK1 signaling.

Osteoporosis, a systemic bone disease that is characterized by a reduction in bone mass and destruction of bone microstructure, is becoming a serious problem worldwide. Bone marrow mesenchymal stem cells (BMSCs) can differentiate into bone-forming osteoblasts, and play an important role in maintaining homeostasis of bone metabolism, thus being a potential therapeutic target for osteoporosis. Although the phytochemical alpinetin (APT) has been reported to possess a variety of pharmacological activities, it is still unclear whether APT can influence the osteogenic differentiation of on BMSCs and if it can improve osteoporosis. In this study, we found that APT treatment was able to enhance osteogenic differentiation levels of human BMSCs in vitro and mouse ones in vivo as revealed by multiple osteogenic markers including increased alkaline phosphatase activity and osteocalcin expression. Mechanistically, the protein kinase A (PKA)/mTOR/ULK1 signaling was involved in the action of APT to enhance the osteogenic differentiation of BMSCs. In addition, oral administration of APT significantly mitigated the bone loss in a dexamethasone-induced mouse model of osteoporosis through strengthening PKA signaling and autophagy. Altogether, these data demonstrate that APT promotes osteogenic differentiation in BMSCs by augmenting the PKA/mTOR/ULK1 autophagy signaling, highlighting its potential therapeutic application for treating osteoporotic diseases.

SMAD-specific E3 ubiquitin ligase 2 promotes angiogenesis by facilitating PTX3 degradation in MSCs from patients with ankylosing spondylitis.

Dysregulated angiogenesis of mesenchymal stem cells (MSCs) is closely related to inflammation and disrupted bone metabolism in patients with various autoimmune diseases. However, the role of MSCs in the development of abnormal angiogenesis in patients with ankylosing spondylitis (AS) remains unclear. In this study, we cultured human umbilical vein endothelial cells (HUVECs) with bone marrow-derived MSCs from patients with AS (ASMSCs) or healthy donors (HDMSCs) in vitro. Then, the cocultured HUVECs were assayed using a cell counting kit-8 (CCK-8) to evaluate the cell proliferation. A wound healing assay was performed to investigate cell migration, and a tube formation assay was conducted to determine the angiogenesis efficiency. ASMSCs exhibited increased angiogenesis, and increased expression of SMAD-specific E3 ubiquitin ligase 2 (Smurf2) in MSCs was the main cause of abnormal angiogenesis in patients with AS. Downregulation of Smurf2 in ASMSCs blocked angiogenesis, whereas overexpression of Smurf2 in HDMSCs promoted angiogenesis. The pro-angiogenic effect of Smurf2 was confirmed by the results of a Matrigel plug assay in vivo. By functioning as an E3 ubiquitin ligase in MSCs, Smurf2 regulated the levels of pentraxin 3 (PTX3), which has been shown to suppress angiogenesis through the PTX3-fibroblast growth factor 2 pathway. Moreover, Smurf2 transcription was regulated by activating transcription factor 4-induced endoplasmic reticulum stress. In conclusion, these results identify novel roles of Smurf2 in negatively regulating PTX3 stability and promoting angiogenesis in ASMSCs.

LncRNA MRF drives the regulatory function on monocyte recruitment and polarization through HNRNPD-MCP1 axis in mesenchymal stem cells.

BACKGROUND: Mesenchymal stem cells (MSCs) exhibit two bidirectional immunomodulatory abilities: proinflammatory and anti-inflammatory regulatory effects. Long noncoding RNAs (lncRNAs) have important functions in the immune system. Previously, we performed high-throughput sequencing comparing lncRNA expression profiles between MSCs cocultured with or without CD14+ monocytes and screened out a new lncRNA termed lncRNA MCP1 regulatory factor (MRF). However, the mechanism of MRF in MSCs is still unknown. METHODS: MRF expression was quantified via qRT-PCR. RNA interference and lentiviruses were used to regulate MRF expression. The immunomodulatory effects of MSCs on monocytes were evaluated via monocyte migration and macrophage polarization assays. RNA pull-down and mass spectrometry were utilized to identify downstream factors of MRF. A dual-luciferase reporter assay was applied to analyze the transcription factors regulating MRF. qRT-PCR, western blotting and ELISAs were used to assess MCP1 expression. A human monocyte adoptive transfer mouse model was applied to verify the function of MRF in vivo. RESULTS: MRF was upregulated in MSCs during coculture with CD14+ monocytes. MRF increased monocyte recruitment by upregulating the expression of monocyte chemotactic protein (MCP1). Knockdown of MRF enhanced the regulatory effect of MSCs on restraining M1 polarization and facilitating M2 polarization. Mechanistically, MRF bound to the downstream protein heterogeneous nuclear ribonucleoprotein D (HNRNPD) to upregulate MCP1 expression, and the transcription factor interferon regulatory factor 1 (IRF1) activated MRF transcription early during coculture. The human monocyte adoptive transfer model showed that MRF downregulation in MSCs inhibited monocyte chemotaxis and enhanced the effects of MSCs to inhibit M1 macrophage polarization and promote M2 polarization in vivo. CONCLUSION: We identified the new lncRNA MRF, which exhibits proinflammatory characteristics. MRF regulates the ability of MSCs to accelerate monocyte recruitment and modulate macrophage polarization through the HNRNPD-MCP1 axis and initiates the proinflammatory regulatory process in MSCs, suggesting that MRF is a potential target to improve the clinical effect of MSC-based therapy or correct MSC-related immunomodulatory dysfunction under pathological conditions.

CCR9 initiates epithelial-mesenchymal transition by activating Wnt/ß-catenin pathways to promote osteosarcoma metastasis.

BACKGROUND: Osteosarcoma (OS) patients with lung metastasis have poor prognoses, and effective therapeutic strategies for delaying or inhibiting the spread of lung metastasis from the primary OS site are lacking. Hence, it is critical to elucidate the underlying mechanisms of OS metastasis and to identify additional new effective treatment strategies for patients. METHODS: Differential expression and functional analyses were performed to identify key genes and relevant signaling pathways associated with OS lung metastasis. The expression of CCR9 in OS cell lines and tissues was measured by RT-qPCR, western blotting and immunohistochemistry. Cell migration and invasion were assessed by wound healing and Transwell Matrigel invasion assays, respectively. The regulatory relationship between CCR9 and the Wnt/ß-catenin signaling pathway was further evaluated by rescue experiments. RESULTS: The expression of CCR9 was elevated in OS cell lines and patients with lung metastasis. CCR9 promoted MG63 and HOS cell migration and invasion by activating the Wnt/ß-catenin signaling pathway. Furthermore, knockdown of CCR9 repressed epithelial-mesenchymal transition (EMT) by downregulating mesenchymal markers (N-cadherin and Vimentin) and EMT-associated transcription factors (twist and snail) and upregulating an epithelial marker (E-cadherin). CONCLUSIONS: Our findings suggest that CCR9 promotes EMT by activating Wnt/ß-catenin pathways to promote OS metastasis. CCR9 may be a promising therapeutic target to inhibit lung metastasis and serve as a novel prognostic marker for OS.

SIRT5-mediated lysine desuccinylation impacts diverse metabolic pathways.

Protein function is regulated by diverse posttranslational modifications. The mitochondrial sirtuin SIRT5 removes malonyl and succinyl moieties from target lysines. The spectrum of protein substrates subject to these modifications is unknown. We report systematic profiling of the mammalian succinylome, identifying 2,565 succinylation sites on 779 proteins. Most of these do not overlap with acetylation sites, suggesting differential regulation of succinylation and acetylation. Our analysis reveals potential impacts of lysine succinylation on enzymes involved in mitochondrial metabolism; e.g., amino acid degradation, the tricarboxylic acid cycle (TCA) cycle, and fatty acid metabolism. Lysine succinylation is also present on cytosolic and nuclear proteins; indeed, we show that a substantial fraction of SIRT5 is extramitochondrial. SIRT5 represses biochemical activity of, and cellular respiration through, two protein complexes identified in our analysis, pyruvate dehydrogenase complex and succinate dehydrogenase. Our data reveal widespread roles for lysine succinylation in regulating metabolism and potentially other cellular functions.

LncRNA-mRNA expression profiles and functional networks in osteoclast differentiation.

Human osteoclasts are differentiated from CD14+ monocytes and are responsible for bone resorption. Long non-coding RNAs (lncRNAs) have been proved to be significantly involved in multiple biologic processes, especially in cell differentiation. However, the effect of lncRNAs in osteoclast differentiation is less appreciated. In our study, RNA sequencing (RNA-seq) was used to identify the expression profiles of lncRNAs and mRNAs in osteoclast differentiation. The results demonstrated that expressions of 1117 lncRNAs and 296 mRNAs were significantly altered after osteoclast differentiation. qRT-PCR assays were performed to confirm the expression profiles, and the results were almost consistent with the RNA-seq data. GO and KEGG analyses were used to predict the functions of these differentially expressed mRNA and lncRNAs. The Path-net analysis demonstrated that MAPK pathway, PI3K-AKT pathway and NF-kappa B pathway played important roles in osteoclast differentiation. Co-expression networks and competing endogenous RNA networks indicated that ENSG00000257764.2-miR-106a-5p-TIMP2 may play a central role in osteoclast differentiation. Our study provides a foundation to further understand the role and underlying mechanism of lncRNAs in osteoclast differentiation, in which many of them could be potential targets for bone metabolic disease.

LncRNA-OG Promotes the Osteogenic Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells Under the Regulation of hnRNPK.

Bone marrow-derived mesenchymal stem cells (BM-MSCs) are the main source of osteoblasts in vivo and are widely used in stem cell therapy. Previously, we analyzed long noncoding RNA (lncRNA) expression profiles during BM-MSC osteogenesis, and further investigation is needed to elucidate how lncRNAs regulate BM-MSC osteogenesis. Herein, we used customized microarrays to determine lncRNA expression profiles in BM-MSCs on days 0 and 10 of osteogenic differentiation. In addition, we identified a novel osteogenesis-associated lncRNA (lncRNA-OG) that is upregulated during this process. Functional assays showed that lncRNA-OG significantly promotes BM-MSC osteogenesis. Mechanistically, lncRNA-OG interacts with heterogeneous nuclear ribonucleoprotein K (hnRNPK) protein to regulate bone morphogenetic protein signaling pathway activation. Surprisingly, hnRNPK positively regulates lncRNA-OG transcriptional activity by promoting H3K27 acetylation of the lncRNA-OG promoter. Therefore, our study revealed a novel lncRNA with a positive function on BM-MSC osteogenic differentiation and proposed a new interaction between hnRNPK and lncRNA. Stem Cells 2018 Stem Cells 2019;37:270-283.

The Relationship of Bone Mineral Density to Oxidant/Antioxidant Status and Inflammatory and Bone Turnover Markers in a Multicenter Cross-Sectional Study of Young Men with Ankylosing Spondylitis.

Low bone mineral density (BMD) is an important complication of ankylosing spondylitis (AS) that seriously affects men and their quality of life, even in young patients. However, the relationships among redox; levels of bone turnover markers (BTMs), inflammatory markers and disease activity; and low BMD in AS require clarification. We recruited 102 men aged 30-39 year with AS and 102 healthy, sex- and age-matched controls for this cross-sectional study. The subjects were analyzed for lumbar spine and femoral neck BMD by dual-energy X-ray absorptiometry. Significantly lower BMD and corresponding T-scores were observed in the AS patients compared with the controls (P < 0.05). The oxidant biomarker and antioxidant levels were significantly (P < 0.05) higher and lower, respectively, in the AS subjects compared with the controls, and the bone resorption and inflammatory marker levels were higher (P < 0.05). In subgroup analyses, the patients with osteoporosis or active disease had the highest levels of oxidant biomarkers (P < 0.05). Furthermore, the BMD T-scores in AS were found to be negatively correlated with oxidative status (P < 0.05). Multivariate binary logistic analysis showed that low BMD in the AS patients was associated with higher levels of advanced oxidation protein products, malondialdehyde and C-terminal telopeptide of type I collagen; lower levels of glutathione peroxidase; and higher scores of a bath ankylosing spondylitis metrology index. In conclusion, imbalanced redox was independently associated with low BMD in young men with AS and may play an important role in the pathogenesis of AS-related low BMD.

Identification of lysine succinylation substrates and the succinylation regulatory enzyme CobB in Escherichia coli.

Lysine succinylation is a newly identified protein post-translational modification pathway present in both prokaryotic and eukaryotic cells. However, succinylation substrates and regulatory enzyme(s) remain largely unknown, hindering the biological study of this modification. Here we report the identification of 2,580 bacterial lysine succinylation sites in 670 proteins and 2,803 lysine acetylation (Kac) sites in 782 proteins, representing the first lysine succinylation dataset and the largest Kac dataset in wild-type E. coli. We quantified dynamic changes of the lysine succinylation and Kac substrates in response to high glucose. Our data showed that high-glucose conditions led to more lysine-succinylated proteins and enhanced the abundance of succinyllysine peptides more significantly than Kac peptides, suggesting that glucose has a more profound effect on succinylation than on acetylation. We further identified CobB, a known Sir2-like bacterial lysine deacetylase, as the first prokaryotic desuccinylation enzyme. The identification of bacterial CobB as a bifunctional enzyme with lysine desuccinylation and deacetylation activities suggests that the eukaryotic Kac-regulatory enzymes may have enzymatic activities on various lysine acylations with very different structures. In addition, it is highly likely that lysine succinylation could have unique and more profound regulatory roles in cellular metabolism relative to lysine acetylation under some physiological conditions.

Lysine succinylation and lysine malonylation in histones.

Histone protein post-translational modifications (PTMs) are significant for gene expression and DNA repair. Here we report the identification and validation of a new type of PTM in histones, lysine succinylation. The identified lysine succinylated histone peptides were verified by MS/MS of synthetic peptides, HPLC co-elution, and isotopic labeling. We identified 13, 7, 10, and 7 histone lysine succinylation sites in HeLa, mouse embryonic fibroblast, Drosophila S2, and Saccharomyces cerevisiae cells, respectively. We demonstrated that this histone PTM is present in all eukaryotic cells we examined. Mutagenesis of succinylation sites followed by functional assays implied that histone lysine succinylation can cause unique functional consequences. We also identified one and two histone lysine malonylation sites in HeLa and S. cerevisiae cells, respectively. Our results therefore increase potential combinatorial diversity of histone PTMs and suggest possible new connections between histone biology and metabolism.

A novel complex networks clustering algorithm based on the core influence of nodes.

In complex networks, cluster structure, identified by the heterogeneity of nodes, has become a common and important topological property. Network clustering methods are thus significant for the study of complex networks. Currently, many typical clustering algorithms have some weakness like inaccuracy and slow convergence. In this paper, we propose a clustering algorithm by calculating the core influence of nodes. The clustering process is a simulation of the process of cluster formation in sociology. The algorithm detects the nodes with core influence through their betweenness centrality, and builds the cluster's core structure by discriminant functions. Next, the algorithm gets the final cluster structure after clustering the rest of the nodes in the network by optimizing method. Experiments on different datasets show that the clustering accuracy of this algorithm is superior to the classical clustering algorithm (Fast-Newman algorithm). It clusters faster and plays a positive role in revealing the real cluster structure of complex networks precisely.

One-Pot Construction of Articular Cartilage-Like Hydrogel Coating for Durable Aqueous Lubrication.

Articular cartilage has an appropriate multilayer structure and superior tribological properties and provides a structural paradigm for design of lubricating materials. However, mimicking articular cartilage traits on prosthetic materials with durable lubrication remains a huge challenge. Herein, an ingenious three-in-one strategy is developed for constructing an articular cartilage-like bilayer hydrogel coating on the surface of ultra-high molecular weight polyethylene (BH-UPE), which makes full use of conceptions of interfacial interlinking, high-entanglement crosslinking, and interface-modulated polymerization. The hydrogel coating is tightly interlinked with UPE substrate through hydrogel-UPE interchain entanglement and bonding. The hydrogel chains are highly entangled with each other to form a dense tough layer with negligible hysteresis for load-bearing by reducing the amounts of crosslinker and hydrophilic initiator to p.p.m. levels. Meanwhile, the polymerization of monomers in the top surface region is suppressed via interface-modulated polymerization, thus introducing a porous surface for effective aqueous lubrication. As a result, BH-UPE exhibits an ultralow friction coefficient of 0.0048 during 10 000 cycles under a load of 0.9 MPa, demonstrating great potential as an advanced bearing material for disc prosthesis. This work may provide a new way to build stable bilayer coatings and have important implications for development of biological lubricating materials.