Aggrecanases that include ADAMTS1, 4, 5, 8, 9 and 15 are considered to play key roles in aggrecan degradation in osteoarthritic cartilage. Here we demonstrate that calcium pentosan polysulfate (CaPPS) directly inhibits the aggrecanase activity of ADAMTS4 without affecting the mRNA expression of the ADAMTS species in interleukin-1alpha-stimulated osteoarthritic chondrocytes. Synthetic peptides corresponding to specific regions of the thrombospondin type 1 repeat, cysteine-rich or spacer domain of ADAMTS4 inhibit the binding to immobilized CaPPS. These data suggest that CaPPS could function as chondroprotective agent for the treatment of osteoarthritis by inhibition of ADAMTS4 through interaction with the C-terminal ancillary domain.

Iron is an essential element in biological systems, but excess iron promotes the formation of reactive oxygen species, resulting in cellular toxicity. Several iron-related genes are highly expressed in the liver, a tissue in which hepatocyte nuclear factor 4α (HNF4α) plays a critical role in controlling gene expression. Therefore, the role of hepatic HNF4α in iron homeostasis was examined using liver-specific HNF4α-null mice (Hnf4a(ΔH) mice). Hnf4a(ΔH) mice exhibit hypoferremia and a significant change in hepatic gene expression. Notably, the expression of transferrin receptor 2 (Tfr2) mRNA was markedly decreased in Hnf4a(ΔH) mice. Promoter analysis of the Tfr2 gene showed that the basal promoter was located at a GC-rich region upstream of the transcription start site, a region that can be transactivated in an HNF4α-independent manner. HNF4α-dependent expression of Tfr2 was mediated by a proximal promoter containing two HNF4α-binding sites located between the transcription start site and the translation start site. Both the GC-rich region of the basal promoter and the HNF4α-binding sites were required for maximal transactivation. Moreover, siRNA knockdown of HNF4α suppressed TFR2 expression in human HCC cells. These results suggest that Tfr2 is a novel target gene for HNF4α, and hepatic HNF4α plays a critical role in iron homeostasis.

TAK-475 (lapaquistat acetate) and its active metabolite-I (TAK-475 M-I) inhibit squalene synthase, which catalyzes the conversion of farnesyl diphosphate (FPP) to squalene. FPP is a substrate for synthesis of other mevalonate-derived isoprenoids (MDIs) such as farnesol (FOH), geranlygeranyl diphosphate (GGPP), and geranylgeraniol. In patients with MKD, a rare autosomal recessive disorder, defective activity of mevalonate kinase leads to a shortage of MDIs. MDIs especially GGPP are required for prenylation of proteins, which is a posttranslation modification necessary for proper functioning of proteins like small guanosine triphosphatases. Malfunction of prenylation of proteins results in upregulation of the inflammatory cascade, leading to increased production of proinflammatory cytokines like interleukin-1β (IL-1β), eventually leading to episodic febrile attacks. In vitro, TAK-475 M-I incubation in a concentration dependent manner increased levels of FPP, GGPP, and FOH in human monocytic THP-1 cells. In subsequent experiments, THP-1 cells or human peripheral blood mononuclear cells (PBMCs) were incubated with simvastatin, which inhibits hydroxymethylglutaryl-coenzyme A reductase and thereby decreases levels of the precursors of MDIs, leading to the depletion of MDIs as expected in MKD patients. Increased levels of GGPP and FPP attenuated lipopolysaccharide (LPS)-induced IL-1β production in THP-1 cells and human PBMCs in statin-treated conditions. The MDIs also significantly reduced the damaged cell ratio in this active MKD-like condition. Moreover, TAK-475 M-I directly inhibited LPS-induced IL-1β production from statin-treated THP-1 cells. These results show anti-inflammatory and cytoprotective effects of MDIs via TAK-475 M-I treatment in statin-treated immune cells, suggesting that possible therapeutic effects of TAK-475 treatment in MKD patients.

SURP domains are exclusively found in splicing-related proteins in all eukaryotes. SF3A1, a component of the U2 snRNP, has two tandem SURP domains, SURP1, and SURP2. SURP2 is permanently associated with a specific short region of SF3A3 within the SF3A protein complex whereas, SURP1 binds to the splicing factor SF1 for recruitment of U2 snRNP to the early spliceosomal complex, from which SF1 is dissociated during complex conversion. Here, we determined the solution structure of the complex of SURP1 and the human SF1 fragment using nuclear magnetic resonance (NMR) methods. SURP1 adopts the canonical topology of α1-α2-310 -α3, in which α1 and α2 are connected by a single glycine residue in a particular backbone conformation, allowing the two α-helices to be fixed at an acute angle. A hydrophobic patch, which is part of the characteristic surface formed by α1 and α2, specifically contacts a hydrophobic cluster on a 16-residue α-helix of the SF1 fragment. Furthermore, whereas only hydrophobic interactions occurred between SURP2 and the SF3A3 fragment, several salt bridges and hydrogen bonds were found between the residues of SURP1 and the SF1 fragment. This finding was confirmed through mutational studies using bio-layer interferometry. The study also revealed that the dissociation constant between SURP1 and the SF1 fragment peptide was approximately 20 μM, indicating a weak or transient interaction. Collectively, these results indicate that the interplay between U2 snRNP and SF1 involves a transient interaction of SURP1, and this transient interaction appears to be common to most SURP domains, except for SURP2.

Protein arginine methyltransferases (PRMTs) regulate diverse biological processes and are increasingly being recognized for their potential as drug targets. Here we report the discovery of a potent, selective, and cell-active chemical probe for PRMT7. SGC3027 is a cell permeable prodrug, which in cells is converted to SGC8158, a potent, SAM-competitive PRMT7 inhibitor. Inhibition or knockout of cellular PRMT7 results in drastically reduced levels of arginine monomethylated HSP70 family stress-associated proteins. Structural and biochemical analyses reveal that PRMT7-driven in vitro methylation of HSP70 at R469 requires an ATP-bound, open conformation of HSP70. In cells, SGC3027 inhibits methylation of both constitutive and inducible forms of HSP70, and leads to decreased tolerance for perturbations of proteostasis including heat shock and proteasome inhibitors. These results demonstrate a role for PRMT7 and arginine methylation in stress response.

We recently reported a unique antioxidant response element (ARE)-activator, BTZO-1, which induced expression of cytoprotective proteins such as heme oxygenase-1 (HO-1) and suppressed oxidative stress-induced cardiomyocyte apoptosis via binding to macrophage migration inhibitory factor (MIF). HO-1 induction and apoptosis inhibition have been reported to improve the outcomes following experimental sepsis by protecting the organs. Therefore, we investigated the potential of BTZO-2, an active BTZO-1 derivative, as a drug for sepsis. BTZO-2 significantly protected mice from the endotoxic shock induced by 5mg/kg lipopolysaccharide (LPS); survival rates increased from 42% to 100%. In contrast, BTZO-2 did not provide significant protection to mice from the shock induced by 10 μg/kg LPS together with d-galactosamine (d-GalN, hepatocyte-specific transcription inhibitor) (LPS/d-GalN). Hepatic HO-1 protein was up-regulated by BTZO-2 in mice injected with 5mg/kg LPS, but not in those injected with 10 μg/kg LPS/d-GalN. Interestingly, BTZO-2 showed little or no effect on LPS-induced up-regulation of plasma cytokine levels in mice. Thus, the organ protection mediated by HO-1 may have a pivotal role in the pharmacological effect of BTZO-2. These results suggest that BTZO-2 is a promising compound for a novel drug for sepsis.

The CIDE (cell death-inducing DFF45-like effector) family composed of CIDEA, CIDEB, CIDEC/FSP27 (fat-specific protein 27), has a critical role in growth of lipid droplets. Of these, CIDEB and CIDEC2/FSP27B are abundant in the liver, and the steatotic livers, respectively. Hepatocyte nuclear factor 4α (HNF4α) has an important role in lipid homeostasis because liver-specific HNF4α-null mice (Hnf4aΔHep mice) exhibit hepatosteatosis. We investigated whether HNF4α directly regulates expression of CIDE family genes. Expression of Cideb and Fsp27b was largely decreased in Hnf4aΔHep mice, while expression of Cidea was increased. Similar results were observed only in CIDEC2, the human orthologue of the Fsp27b, in human hepatoma cell lines in which HNF4α expression was knocked down. Conversely, overexpression of HNF4α strongly induced CIDEC2 expression in hepatoma cell lines. Furthermore, HNF4α transactivated Fsp27b by direct binding to an HNF4α response element in the Fsp27b promoter. In addition, Fsp27b is known to be transactivated by CREBH that is regulated by HNF4α, and expression of CREBH was induced by HNF4α in human hepatoma cells. Co-transfection of HNF4α and CREBH resulted in synergistic transactivation and induction of Fsp27b compared to that of HNF4α or CREBH alone. These results suggest that HNF4α, in conjunction with CREBH, plays an important role in regulation of Fsp27b expression.