Tip60 Phosphorylation at Ser 99 Is Essential for Autophagy Induction in Bombyx mori

Tip60, a key histone acetyltransferase of the MYST family and member of the nuclear multimeric protein complex (NuA4), regulates the activity and stability of proteins involved in the cell cycle, DNA damage responses, autophagy, etc. However, the function and regulatory mechanism of Tip60 homolog in Bombyx mori are not elucidated. In the present study, Bombyx Tip60 (BmTip60) was functionally identified. Developmental profiles showed that the protein levels and nuclear localization of BmTip60 peaked in fat body during the larval–pupal metamorphosis when autophagy was intensive; simultaneously, the BmTip60 protein migrated to form an upper band as detected by Western blot. Interestingly, the upper band of BmTip60 was reduced by λ-phosphatase treatment, indicating that it was a phosphorylated form of BmTip60. Results showed that BmTip60 was promoted by starvation but not 20-hydroxyecdysone treatment. Transcription factor AMP-activated protein kinase (AMPK) affected by starvation was pivotal for BmTip60 protein migration. In addition, one mammalian phosphorylation site was identified in BmTip60 at Ser99, the constitutive-activation mutation of Ser99 to Asp99 but not its inactive mutation to Ala99 significantly upregulated autophagy, showing the critical role of phosphorylation at Ser99 for BmTip60-mediated autophagy. In conclusion, the starvation-AMPK axis promotes BmTip60 in B. mori, which was requisite for autophagy induction. These results reveal a regulatory mechanism of histone acetyltransferase Tip60 homologs by phosphorylation in insects, and sheds light on further related studies of acetylation regulation.

Proteomic Analysis of Lipid Droplets in Sesamum indicum

We attempted to identify the total proteome in sesame lipid droplets. Results from two-dimensional electrophoresis showed 139 protein spots in lipid droplet samples. Each spot was isolated, digested with trypsin, and applied to liquid chromatography–tandem mass spectrometry (Q-Tof Premier). As a result, 103 spots were identified. Although oleosin, caleosin, and steroleosin are known major components of the lipid droplet, many other proteins were also found in the lipid droplet. In addition to the three major proteins, TAG factor protein, glyceraldehyde-3-phosphate dehydrogenase, F1 ATPase, 70-kDa heat shock protein, seed maturation protein PM24, and 11S globulin precursor isoforms 3 and 4 were found in the lipid droplet. Three types of oleosins, 15-, 15.5-, and 17-kDa were present in the sesame lipid droplet, and the 15.5-kDa oleosin had high homology with oleosin from Coffea canephora. It has been shown by acid phosphatase treatment that oleosin proteins contain phosphate groups. Protein disulfide-isomerase 2 precursor, calreticulin-1, and BiP, which are known as marker proteins of the endoplasmic reticulum, were found as the components of the lipid droplet. Immunoconfocal microscopy was used to show that 11S globulin precursor isoform 3 and 4 were indeed localized in the lipid droplet. The presence of 11S globulin in the lipid droplets suggested a new mechanism for the lipid droplet formation.

Phosphorylation and SCF-mediated degradation regulate CREB-H transcription of metabolic targets

CREB‑H, an endoplasmic reticulum–anchored transcription factor, plays a key role in regulating secretion and in metabolic and inflammatory pathways, but how its activity is modulated remains unclear. We examined processing of the nuclear active form and identified a motif around S87–S90 with homology to DSG-type phosphodegrons. We show that this region is subject to multiple phosphorylations, which regulate CREB-H stability by targeting it to the SCFFbw1aE3 ubiquitin ligase. Data from phosphatase treatment, use of phosophospecific antibody, and substitution of serine residues demonstrate phosphorylation of candidate serines in the region, with the core S87/S90 motif representing a critical determinant promoting proteasome-mediated degradation. Candidate kinases CKII and GSK-3b phosphorylate CREB-H in vitro with specificities for different serines. Prior phosphorylation with GSK-3 at one or more of the adjacent serines substantially increases S87/S90-dependent phosphorylation by CKII. In vivo expression of a dominant-negative Cul1 enhances steady-state levels of CREB‑H, an effect augmented by Fbw1a. CREB-H directly interacts with Fbw1a in a phosphorylation-dependent manner. Finally, mutations within the phosphodegron, when incorporated into the full-length protein, result in increased levels of constitutively cleaved nuclear protein and increased transcription and secretion of a key endogenous target gene, apolipoprotein A IV.

Hepatoprotective and Antioxidant Activities of Zizphus rotundifolia (Linn.) against Carbon Tetrachloride-Induced Hepatic Damage in Rats

Many hepatoprotective herbal preparations have been recommended in alternative system of medicine for the treatment of hepatic disorders. There are few or no systemic studies have been done on protective efficacy of Ziziphus rotundifolia (Rhamnaceae) to treat liver diseases. In this study, hepatoprotective and antioxidant activity of ethanolic extracts was evaluated against CCl4-induced liver damage in rats. Liver damage was evidenced by elevated levels of biochemical parameters such as serum glutamate oxaloacetic acid transaminase, glutamate pyruvic transaminase and serum alkaline phosphatase. Treatment with ethanolic extracts of Z. rotundifolia (300 mg/kg, p.o.) produced a significant reversal in the above biochemical parameters, and reducing power, superoxide anion scavenging activity and reduced histopathological scores. These findings suggest that the extracts of Zizphus rotundifolia possess significant hepatoprotective and antioxidant properties.

Polyubiquitin Binding to Optineurin Is Required for Optimal Activation of TANK-binding Kinase 1 and Production of Interferon β

TANK-binding kinase (TBK1) is essential for transcription of the interferon (IFN) β gene in response to lipopolysaccharide (LPS) and double-stranded RNA, but the molecular mechanisms that underlie the activation of TBK1 are incompletely understood. Previously, we identified the NF-κB essential modulator (NEMO)-related polyubiquitin-binding protein, optineurin (OPTN), as a novel binding partner of TBK1. To determine whether the ubiquitin-binding function of OPTN is involved in regulating TBK1 and IFNβ production, we generated a mouse in which wild-type optineurin was replaced by the polyubiquitin binding-defective mutant, OPTND477N/D477N. In this study, we found that LPS or poly(I:C)-induced TBK1 activity was significantly reduced in bone marrow-derived macrophage (BMDM) from OPTND477N/D477N mice. Consistent with this, the phosphorylation of IFN regulatory factor 3 (IRF3) and the production of IFNβ mRNA and secretion were reduced. Stimulation of BMDMs with LPS triggered the phosphorylation of OPTN, which was reversed by phosphatase treatment and prevented by pharmacological inhibition of both the canonical IκB kinases (IKKα/β) and the IKK-related kinases (TBK1/IKKϵ). In contrast, LPS-stimulated phosphorylation of OPTN(D477N) was markedly reduced in BMDMs from OPTND477N/D477N mice, and inhibition of the canonical IKKs alone prevented phosphorylation, providing further evidence that ubiquitin binding to OPTN contributes to LPS-induced TBK1 activation. TBK1 and IKKβ phosphorylated OPTN preferentially at Ser-177 and Ser-513, respectively, in vitro. In conclusion, our results suggest that OPTN binds to polyubiquitylated species formed in response to LPS and poly(I:C), enhancing the activation of TBK1 that is required for optimal phosphorylation of IRF3 and production of IFNβ.

Hyperphosphorylated paratarg-7: a new molecularly defined risk factor for monoclonal gammopathy of undetermined significance of the IgM type and Waldenström macroglobulinemia

We recently described paratarg-7 (P-7), a protein of unknown function, as the target of 15% of immunoglobulin A (IgA) and IgG paraproteins in monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma. To determine the frequency of P-7 as a paraprotein target in IgM-MGUS and Waldenström macroglobulinemia (WM), sera from patients with IgM-MGUS/WM were tested for reactivity with recombinant P-7 by enzyme-linked immunoabsorbent assay. The specificity of the paraprotein-mediated reaction was shown by absorption studies and cloning of the respective B-cell receptor. The paraproteins of 18 (9 WM and 9 IgM-MGUS) of 161 patients (11%) reacted with P-7. Isoelectric focusing and phosphatase treatment showed that P-7 was hyperphosphorylated (pP-7) in all patients with an anti–P-7-specific IgM paraprotein tested. Because only 4 of 200 healthy controls (2%) were carriers of pP-7, pP-7 carrier state is associated with a significantly increased risk (odds ratio = 6.2; P = .001) for developing IgM-MGUS/MW. Family analyses showed that the pP-7 carrier state is inherited as a dominant trait. After IgA/IgG-MGUS and multiple myeloma, IgM-MGUS/WM is the second neoplasia associated with pP-7 carrier state. The dominant inheritance of pP-7 explains cases of familial IgM-MGUS/WM and enables the identification of family members at increased risk.

Hyperphosphorylation of Antigenic Targets of Paraproteins In MGUS and Multiple Myeloma (MM) Is a Consistent Finding: Implications for the Pathogenesis of MGUS/MM

Abstract 4085 Background: Hyperphosphorylated paratarg-7 (pP-7) is the target of paraproteins in 15% of patients with MGUS/MM (Preuss et al. Int J Cancer 2009; 125:656-61). Carriers of pP-7 have an 8-times increased risk to develop IgA/IgG-MGUS and multiple myeloma (Grass et al., Lancet Oncology 2009; 10:950-956) and a 6.5 times increased risk to develop IgM-MGUS and Waldenström′s macroglobulinemia (Grass et al., Blood 2009;114:1513s). Analysis of affected families showed that pP-7 is inherited in a dominant fashion. Thus, pP-7 is the first molecularly defined autosomal-dominant risk factor for any hematological neoplasm. Objective: Since paratarg-7, a frequent target of paraproteins in patients with MGUS/MM is hyperphosphorylated and inherited in a dominant fashion we extended our studies to investigate the prevalence of further antigenic targets and their phosphorylation state. Methods: Sera of MGUS/MM patients and relatives of affected families were tested for antibody reactivity against antigens represented in a fetal brain derived protein macroarray using a modified SEREX approach (Preuss et al. International J. Cancer 2009;125, 656–661). Lysates of peripheral blood from patients, family-members and controls were tested by gel electrophoresis and isoelectric focusing before and after phosphatase treatment. Results: Analysis of 3 families with multiple members affected by MGUS and MM revealed that the antigenic targets of paraproteins from affected members of a given family are identical: 2 families shared paratarg-7 and 1 family shared paratarg-8 as the family-typic paraprotein antigen. Paratarg-8 is encoded by the ATG13 gene, a member of the „autophagy regulatory complex“ family of genes. Paratarg-8 showed no mutations or polymorphisms in patients compared to controls. However, IEF before and after phosphatase treatment showed that paratarg-8 is hyperphosphorylated (pP-8) in the affected family members compared to healthy controls and that pP-8 carrier state is also inherited in a dominant fashion. This finding prompted us to check previously identified paraprotein targets for hyperphosphorylation, which, in addition to paratarg-7 and paratarg-8 was possible for paratarg 2, 5, 6, 9, 10, 11. In all 8/8 cases, IEF before and after phosphatase treatment revealed that the patients were carriers of a hyperphosphorylated version of their paraprotein target compared to healthy controls. Conclusions: The paraproteins of members affected by familial MGUS/MM are directed against family-specific antigenic targets, suggesting that the genetic background shared by these patients contains a chronic auto-immune response. Paratarg-7 and paratarg-8 are the first family-typic antigens that have been molecularly defined to date. The fact that all antigenic targets of paraproteins molecularly defined to date are hyperphosphorylated in patients compared to healthy controls implies a significant role of the hyperphosphorylation state in these neoplasms. Analysis of the T-cell response against normo- and hyperphosphorylated paraprotein targets in affected and non-affected family members is underway as are genome-wide association to determine the SNP or mutation which is associated with the hyperphosphorylation of paraprotein targets. Disclosures: Lynch: State of Nebraska LB595 support: Research Funding; Charles F. and Mary C. Heider Chair at Creighton University.: Research Funding.

Hepatoprotection by carotenoids in isoniazid–rifampicin induced hepatic injury in rats

This study evaluates the hepatoprotective effect of carotenoids against isoniazid (INH) and rifampicin (RIF). Thirty-six adult rats were divided into the following 4 groups: (1) control group treated with normal saline; (2) INH + RIF group treated with 50 mg·(kg body mass)–1·day–1 of INH and RIF each; (3) INH + RIF+ carotenoids group treated with 50 mg·(kg body mass)–1·day–1 of INH and RIF each and 10 mg·(kg body mass)–1·day–1 of carotenoids; and (4) carotenoids group treated with 10 mg·(kg body mass)–1·day–1 of carotenoids for 28 days intragastrically. Oxidative stress and antioxidant levels in liver and blood, liver histology and change in transaminases were measured in all the above-mentioned groups. There was an increase in lipid peroxidation with a reduction in thiols, catalase, and superoxide dismutase (SOD) in the liver and blood of rats accompanied by an increase in transaminases, bilirubin, and alkaline phosphatase. Treatment with carotenoids along with INH + RIF partially reversed lipid peroxidation, thiols, catalase, and SOD in the liver and blood of rats. Elevated levels of the enzymes in serum were also reversed partially by this treatment. The degree of necrosis, portal triaditis, and inflammation were also lowered in the carotenoids group. In conclusion, carotenoids supplementation in INH + RIF treated rats showed partial protection.