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Autophagy receptor p62/SQSTM1 promotes the assembly and removal of ubiquitylated proteins by forming p62 bodies and mediating their encapsulation in autophagosomes. Here we show that under nutrient-deficient conditions, cellular p62 specifically undergoes acetylation, which is required for the formation and subsequent autophagic clearance of p62 bodies. We identify K420 and K435 in the UBA domain as the main acetylation sites, and TIP60 and HDAC6 as the acetyltransferase and deacetylase. Mechanically, acetylation at both K420 and K435 sites enhances p62 binding to ubiquitin by disrupting UBA dimerization, while K435 acetylation also directly increases the UBA-ubiquitin affinity. Furthermore, we show that acetylation of p62 facilitates polyubiquitin chain-induced p62 phase separation. Our results suggest an essential role of p62 acetylation in the selective degradation of ubiquitylated proteins in cells under nutrient stress, by specifically regulating the assembly of p62 bodies., The autophagy receptor p62 mediates the assembly and removal of ubiquitylated protein aggregates by forming p62 bodies. Here, the authors identify an acetylation-dependent mechanism that regulates formation and autophagic clearance of p62 bodies under nutrient-deficient conditions.

Objectives α2-adrenergic receptors are reportedly involved in cancer cell proliferation, invasion, and apoptosis through regulation of diverse molecules, which implies that it contributes to tumor progression. However, the functional significance of α2-adrenergic receptors in osteosarcoma (OS) is unclear. Tizanidine hydrochloride (THC), an α2-adrenergic receptor agonist, is often used to alleviate symptoms of spasticity. This study investigated the functional implications of THC treatment on human OS cells and the underlying mechanisms of resulting changes. Methods The proliferation of U2 OS cells was assessed by Cell Counting Kit-8; the migration and invasion capacities of U2 OS cells were then analyzed by transwell assay. Moreover, apoptosis in U2 OS cells was evaluated by flow cytometry and western blot analyses. Additionally, expression levels of key proteins in the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) signaling pathway were measured. Results THC inhibited the proliferation, migration, and invasion of U2 OS cells, but promoted apoptosis within these cells. Expression levels of p-AKT, p-mTOR, and p-P70S6K were reduced by exposure to THC, suggesting involvement of PI3K/AKT signaling in THC-induced cytotoxicity within OS cells. Conclusions THC may play a novel role in OS cell cytotoxicity, and these findings suggest a potent therapeutic strategy for OS treatment.

Lanthanoid pseudo-contact shift (PCS) provides long-range structural information between a paramagnetic tag and protein nuclei. However, for proteins with native cysteines, site-specific attachment may only utilize functional groups orthogonal to sulfhydryl chemistry. Here we report two lanthanoid probes, DTTA-C3-yne and DTTA-C4-yne, which can be conjugated to an unnatural amino acid pAzF in the target protein via azide-alkyne cycloaddition. Demonstrated with ubiquitin and cysteine-containing enzyme EIIB, we show that large PCSs of distinct profiles can be generated for each tag/lanthanoid combination. The DTTA-based lanthanoid tags are associated with large magnetic susceptibility tensors owing to the rigidity of the tags. In particular, introduction of the DTTA-C3 tag affords intermolecular PCSs and enables structural characterization of a transient protein complex between ubiquitin and a UBA domain. Together, we have expanded the repertoire of paramagnetic tags and the applicability of paramagnetic NMR.

To investigate the joint health status of patients with hemophilia treated on-demand and to analyze the incidence trend of hemophilic arthritis, so as to probide the scientific clinical data for furture study.The clinical data of patients with hemophilia admitlted in Tianjin municipal first central hospital form March 2016 to October 2017 were collected, the basic information of patients was recorded; the joint health status was evaluated by using the hemophillia Joint Health Score (HJHS) 2.1; the life guality of patients was analyzed by using the MOS item short form-36 health survey, SF-36; the correlation of joint function with life guatity was analyzed by pearson correlation test.196 ont of 210 patients with hemophila were treated on demand. The average age of patients was 27.81(2-73) years old, Among 196 patients, 189 was hemophilia A (96.43%) and 9 was hemophilia B (3.57). The patients without joint involvement, patients with 1 joint and 32 jionts involvement accounted for 3.57%, 11.72% and 84.71% respectively. The incidonce of involvement in elbow, knae and ankle joints was 71.93%, 80.61% and 82.91% respectively. The joint invlvement rate in patients with mild, intermediate and severe hemphilia accounted for 61.66%, 72.40% and 80.73% respectively. The mean HJHS in intermediate and severe henophilia patients was 23.59±17.02 scores and 26.69±17.68 scores respectively, there was no statistical difference (P0.05). The joint fanction in hemophilia patients negatively correlated with life gnality of patients (r = 0.076).The incidence of arthritis in hemophilia patients is high, and at least 1 joint has been involved in patients aged over 10 years old, moreover the multiple joint involvement exrsts in most patients. The impairment of joint function affects the routine behavious and activities.

Specific cell surface labeling is essential for visualizing the internalization processes of G-protein coupled receptors (GPCRs) and for gaining mechanistic insight of GPCR functions. Here we present a rapid, specific and versatile labeling scheme for GPCRs at living-cell membrane with the use of a split green fluorescent protein (GFP). Demonstrated with two GPCRs, GPR17 and CysLT2R, we show that two β-stands (β-stands 10 and 11) derived from a superfolder GFP (sfGFP) can be engineered to one of the three extracellular loop of a GPCR. The complementary fragment of sfGFP has nine β-strands (β-stands 1-9) that carries the mature fluorophore and can be proteolytically derived from the full-length sfGFP. Separately the GFP fragments are non-fluorescent, but become fluorescent upon assembly, thus allowing specific labeling of the target proteins. The two GFP fragments rapidly assemble and the resulting complex is extremely tight under non-denaturing conditions, which allows real-time and quantitative assessment of the internalized GPCRs. We envision that this labeling scheme will be of great use for labeling other membrane proteins in various biological and pharmacological applications.

A polyubiquitin comprises multiple covalently linked ubiquitins and recognizes myriad targets. Free or bound to ligands, polyubiquitins are found in different arrangements of ubiquitin subunits. To understand the structural basis for polyubiquitin quaternary plasticity and to explore the target recognition mechanism, we characterize the conformational space of Lys63-linked diubiquitin (K63-Ub2). Refining against inter-subunit paramagnetic NMR data, we show that free K63-Ub2 exists as a dynamic ensemble comprising multiple closed and open quaternary states. The quaternary dynamics enables K63-Ub2 to be specifically recognized in a variety of signaling pathways. When binding to a target protein, one of the preexisting quaternary states is selected and stabilized. A point mutation that shifts the equilibrium between the different states modulates the binding affinities towards K63-Ub2 ligands. This conformational selection mechanism at the quaternary level may be used by polyubiquitins of different lengths and linkages for target recognition. DOI: http://dx.doi.org/10.7554/eLife.05767.001, eLife digest Proteins can be tagged with other molecules that indicate what the cell should do with that protein. For example, proteins tagged with a small protein called ubiquitin—which is linked to other ubiquitin molecules to form ‘polyubiquitin’—may be destroyed or relocated within a cell. Like all proteins, a ubiquitin is made up of chains of amino acids. Specific amino acids form the linkages between individual ubiquitins to form a polyubiquitin, and the nature of these linkages influences the effect that a polyubiquitin has on the tagged protein. One linkage involves a lysine amino acid at position 63 (known as Lys63). This linkage is found in the polyubiquitin that is involved in repairing damaged proteins and relocating target proteins to a part of the cell where they are utilized for immune response. To perform these different roles, the polyubiquitin must be able to distinguish between a variety of target proteins. The shape that a protein takes on determines how it works, and most proteins constantly and rapidly switch between different shapes. Previous work suggested that the Lys63-linked polyubiquitin could only take on an elongated ‘open’ structure by itself. It was not clear whether the protein could take on a compact ‘closed’ structure without first binding to a target protein. Liu et al. used a technique known as nuclear magnetic resonance (NMR) to explore the high-resolution structures of the Lys63-linked ubiquitin chain when they are not bound to other proteins. The results showed that a large percentage of the protein was in a closed state, and that there were at least one open shape and two kinds of closed shapes. Liu et al. suggest that the shape of the unbound Lys63-linked ubiquitin chain determines what other proteins can be bound, and that the binding stabilizes the shape of the ubiquitin. This mechanism of binding is known as conformational selection. Further work is required to analyze whether other polyubiquitin chains recognize their partners in a similar manner. DOI: http://dx.doi.org/10.7554/eLife.05767.002

B cell translocation gene 2 (BTG2) has been reported to be a potential tumor suppressor in many types of tumors. However, the roles and molecular mechanisms of BTG2 in osteosarcoma progression are still unknown. In this study, we investigated the role of BTG2 in proliferation and metastasis of osteosarcoma and the underlying mechanism. BTG2 expression levels were measured in fresh osteosarcoma tissues and cell lines. The effects of BTG2 on cell proliferation, migration and invasion were explored by MTT, transwell assays, western blot, and in vivo tumorigenesis in nude mice. We found that BTG2 was down-regulated in human osteosarcoma tissues and cell lines. Overexpression of BTG2 inhibited the proliferation and migration/invasion of human osteosarcoma cells in vitro, it also markedly inhibited xenograft tumor growth in vivo. Furthermore, BTG2 significantly decreased the expression of phosphorylated PI3K and AKT in osteosarcoma cells. Taken together, our data indicate that BTG2 might suppress the tumor growth and metastasis via PI3K/AKT signaling pathway, implying that BTG2 may serve as a potential molecular target for the treatment of osteosarcoma.

To establish a method for screening nicotinamide phosphoribosyl transferase (NAMPT) inhibitors based on endogenous fluorescence determination.The double mutants of NAMPT, G355C/D393C, was cross-linked by using 1, 4-Bismaleimidobutane (BMB) to block the entrance of enzymatic active site of NAMPT. The binding of compounds to NAMPT was evaluated according to the change of spontaneous fluorescence of NAMPT and BMB-NAMPT with 280 nm excitation and 333 nm emmision. The in vitro enzamatic activity of NAMPT was determined by nuclear magnetic resonance. The cell viability was determined by MTT assay.FK866 significantly decreased the spontaneous fluorescence of NAMPT but not of BMB-NAMPT. Rosmaric, cynarine and 1, 3-dicaffeoylquinic acid also decreased the spontaneous fluorescence of both NAMPT and BMB-NAMPT. However, the inhibition on two proteins was equivalent. FK866 significantly inhibit the catalysis of NAMPT. Rosmarinic acid, cynarine and 1, 3-dicaffeoylquinic acid failed to inhibit the catalysis of NAMPT. FK866 inhibited the viability of A549 cells, but rosmarinic acid, cynarine and 1, 3-dicaffeoylquinic acid did not.Endogenous fluorescence spectrometry based on NAMPT and BMB-NAMPT protein can be used for screening compounds that bind with NAMPT, and distinguishing the binding site - either within the enzymatic active site or not. Rosmarinic acid, cynarine and 1, 3-dicoffeoylquinic acid can bind to NAMPT out its enzymatic active site.

To analyze the reliability of magnetic resonance imaging (MRI) in detecting posterior ligament complex injury in thoracolumbar fractures.Ninety-five patients with thoracolumbar fracture were evaluated by palpation of the inter-spinal gap, plain radiography, and MRI before operation. In addition to conventional MRI sequences, a fat-suppressed T2-weighted sagittal sequence was performed. Surgery was performed by a posterior approach. During the operation, posterior ligament complex was examined.A wide inter-spinal gap was palpated in 41 patients and was found in 55 patients on plain radiography. According to MRI, injury to the supraspinal ligament was suspected in 85 patients, the inter-spinal ligament in 83 patients, and the ligamentum flavum in 26 patients. There were 82 supraspinal ligament injuries, 80 inter-spinal ligament injuries, and 20 ligamentum flavum injuries in operative findings. The relations between plain radiography and operative findings, between MRI interpretation and operative findings were statistically significant.A fat-suppressed T2-weighted sagittal sequence of MRI is a highly sensitive, specific, and accurate method to detect posterior ligament complex injury and which is recommended for the accurate evaluation of posterior ligament complex injury in thoracolumbar fractures.

To investigate the feasibility that transforming growth factor-beta1 (TGF-beta1) -loaded fibrin sealant (FS) promotes bone marrow mesenchymal stem cells (BMSCs) to create tissue engineering cartilage in vivo.The BMSCs were isolated from healthy human and amplified in vitro, and then induced by defined medium containing TGF-beta1 and dexamethasone. After 7 days the induced BMSCs were collected and mixed with TGF-beta1-loaded FS or FS as BMSCs+ FS-TGF-beta1 group and BMSCs+ FS experimental group. Then the mixture was injected by a needle into the dorsum of nude mice. In control group, only FS or BMSCs were injected. The tissue engineering specimens were harvested from nude mice 12 weeks later. Gross observation, average wet weight measurement, glycosaminoglycan (GAG) quantification, histology and immunohistochemistry were used to evaluate the results.The BMSCs have possessed the shape and functional characters of chondrocyte when transferred to a defined medium. After injection of the mixture, the cartilage-like tissue were formed in two experimental groups. Compared with BMSC+ FS group, the specimens of BMSCs +FS-TGF-beta1 group were larger and firmer. Alcian staining showed better metachromatic matrix formation. The GAG contents were significantly higher. Immunohistochemical staining of collagen type II was stronger. However, no cartilage-like tissue was formed in two control groups.TGF-beta1-loaded FS can promote BMSCs to contract injectable tissue engineering cartilage in vivo.