Proteasome activation as a novel antiaging strategy

Homeostasis is a key feature of cellular lifespan. Maintenance of cellular homeostasis influences the rate of aging and is determined by several factors, including efficient proteolysis of damaged proteins. Protein degradation is predominantly catalyzed by the proteasome. Specifically, the proteasome is responsible for cell clearance of abnormal, denatured or in general damaged proteins as well as for the regulated degradation of short-lived proteins. As proteasome has an impaired function during aging, emphasis has been given recently in identifying ways of its activation. A number of studies have shown that the proteasome can be activated by genetic manipulations as well as by factors that affect its conformation and stability. Importantly the developed proteasome activated cell lines exhibit an extended lifespan. This review article discusses in details the various factors that are involved in proteasome biosynthesis and assembly and how they contribute to its activation. Finally as few natural compounds have been identified having proteasome activation properties, we discuss the advantages of this novel antiaging strategy.     

Ovarian Carcinoma Subtypes Are Different Diseases: Implications for Biomarker Studies

Background

Although it has long been appreciated that ovarian carcinoma subtypes (serous, clear cell, endometrioid, and mucinous) are associated with different natural histories, most ovarian carcinoma biomarker studies and current treatment protocols for women with this disease are not subtype specific. With the emergence of high-throughput molecular techniques, distinct pathogenetic pathways have been identified in these subtypes. We examined variation in biomarker expression rates between subtypes, and how this influences correlations between biomarker expression and stage at diagnosis or prognosis.

Methods and Findings

In this retrospective study we assessed the protein expression of 21 candidate tissue-based biomarkers (CA125, CRABP-II, EpCam, ER, F-Spondin, HE4, IGF2, K-Cadherin, Ki-67, KISS1, Matriptase, Mesothelin, MIF, MMP7, p21, p53, PAX8, PR, SLPI, TROP2, WT1) in a population-based cohort of 500 ovarian carcinomas that was collected over the period from 1984 to 2000. The expression of 20 of the 21 biomarkers differs significantly between subtypes, but does not vary across stage within each subtype. Survival analyses show that nine of the 21 biomarkers are prognostic indicators in the entire cohort but when analyzed by subtype only three remain prognostic indicators in the high-grade serous and none in the clear cell subtype. For example, tumor proliferation, as assessed by Ki-67 staining, varies markedly between different subtypes and is an unfavourable prognostic marker in the entire cohort (risk ratio [RR] 1.7, 95% confidence interval [CI] 1.2%–2.4%) but is not of prognostic significance within any subtype. Prognostic associations can even show an inverse correlation within the entire cohort, when compared to a specific subtype. For example, WT1 is more frequently expressed in high-grade serous carcinomas, an aggressive subtype, and is an unfavourable prognostic marker within the entire cohort of ovarian carcinomas (RR 1.7, 95% CI 1.2%–2.3%), but is a favourable prognostic marker within the high-grade serous subtype (RR 0.5, 95% CI 0.3%–0.8%).

Conclusions

The association of biomarker expression with survival varies substantially between subtypes, and can easily be overlooked in whole cohort analyses. To avoid this effect, each subtype within a cohort should be analyzed discretely. Ovarian carcinoma subtypes are different diseases, and these differences should be reflected in clinical research study design and ultimately in the management of ovarian carcinoma.     

Ontogeny of angiopoietin‐like protein 1, 2, 3, 4, 5, and 7 genes during chick embryonic development

Angiopoietin‐like proteins (ANGPTLs) are secreted proteins possessing an amino‐terminal coiled‐coil domain and a carboxyl‐terminal fibrinogen‐like domain and are known as angiogenic factors. Several members of ANGPTLs also regulate lipid metabolism independently of angiogenic effects, but most of their functions during vertebrate development are not demonstrated. To ascertain their developmental functions, we examined the expression patterns of Angptl1, 2, 3, 4, 5, and 7 orthologues during chick development using whole‐mount in situ hybridization. Angptl1 was first detected at embryonic day 3 (E3) in the somite. At E4, Angptl1 was expressed in somite‐derivatives and limb mesenchyme. Angptl2 was first detected at E3 in the hindbrain. At E4, Angptl2 was expressed in neuroepithelium of forebrain and hindbrain and partly in the heart. Angptl3 was first detected at E3 and continued to be expressed in the liver and yolk sac at E4. Angptl4 was first detected at E3 in the somites and liver. At E4, Angptl4 was also observed in the heart. Angptl5 was not detected in these developmental stages. Angptl7 was first detected at E3 in the ectoderm overlying the lenses of the eyes. At E4, Angptl7 was specifically expressed in cornea. These data suggest that each member of the ANGPTL family could be related to angiogenesis during various organogeneses of the developing chick embryo.     

Capillary electrophoresis-electrospray ionization mass spectrometry for rapid and sensitive N-glycan analysis of glycoproteins as 9-fluorenylmethyl derivatives

It is well known that most protein therapeutics such as monoclonal antibody pharmaceuticals and other biopharmaceuticals including cancer biomarkers are glycoproteins, and thus the development of high-throughput and sensitive analytical methods for glycans is essential in terms of their determination and quality control. We previously reported a novel alternative labeling method for glycans involving 9-fluorenylmethyl chloroformate (Fmoc-Cl) instead of the conventional reductive amination procedure. The derivatives were analyzed by high-performance liquid chromatography (HPLC) (Kamoda S, Nakano M, Ishikawa R, Suzuki S, Kakehi K. 2005. Rapid and sensitive screening of N-glycans as 9-fluorenylmethyl derivatives by high-performance liquid chromatography: A method which can recover free oligosaccharides after analysis. J Proteome Res. 4:146-152). This method was rapid and simple; however, it was time-consuming in terms of analysis by HPLC and did not provide so much information such as the detailed structures and mass numbers of glycans. Here we have developed a high-throughput and highly sensitive method. It comprises three steps, i.e., release of glycans, derivatization with Fmoc, and capillary electrophoresis-electrospray ionization mass spectrometry (CE-ESI MS) analysis. We analyzed several glycoproteins such as fetuin, alpha1 acid glycoprotein, IgG, and transferrin in order to validate this method. We were able to analyze the above glycoproteins with the three-step procedure within only 5 h, which provided detailed N-glycan patterns. Moreover, the MS/MS analysis allowed identification of the N-glycan structures. As novel applications, the method was employed for the analysis of N-glycans derived from monoclonal antibody pharmaceuticals and also from alpha-fetoprotein; the latter is known as one of the tumor markers of hepatocellular carcinomas. We were able to easily and rapidly determine the detailed structures of the N-glycans. The present method is very useful for the analysis of large numbers of samples such as a routine analysis.     

Glucose-induced production of hydrogen sulfide may protect the pancreatic beta-cells from apoptotic cell death by high glucose

We examined the expression of the major H₂S-producing enzymes, cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE). CBS was ubiquitously distributed in the mouse pancreas, but CSE was found only in the exocrine. Freshly isolated islets expressed CBS, while CSE was faint. However, high glucose increased the CSE expression in the beta-cells. l-Cysteine or NaHS suppressed islet cell apoptosis with high glucose, and increased glutathione content in MIN6 beta-cells. Pretreatment with l-cysteine improved the secretory responsiveness following stimulation with glucose. The CSE inhibitor dl-propargylglycine antagonized these l-cysteine effects. We suggest H₂ S may function as an ‘intrinsic brake’ which protects beta-cells from glucotoxicity.     

Study on the compounds containing 19F and 10B atoms in a single molecule for the application to MRI and BNCT

Magnetic resonance imaging (MRI) and boron-neutron capture therapy (BNCT) are quite attractive techniques for diagnosis and treatment of cancer, respectively. In order to progress the study on both MRI and BNCT, the novel compounds containing 19F and 10B atoms in a single molecule were designed and synthesized. In the present paper, the syntheses and the internalization rates into tumor cells of these compounds are elucidated.     

Free radicals impair the anti-oxidative stress activity of DJ-1 through the formation of SDS-resistant dimer

DJ-1 is a causative gene for familial Parkinson’s disease (PD). Loss-of-function of DJ-1 protein is suggested to contribute to the onset of PD, but the causes of DJ-1 dysfunction remain insufficiently elucidated. In this study, we found that the SDS-resistant irreversible dimer of DJ-1 protein was formed in human dopaminergic neuroblastoma SH-SY5Y cells when the cells were exposed to massive superoxide inducers such as paraquat and diquat. The dimer was also formed in vitro by superoxide in PQ redox cycling system and hydroxyl radical produced in Fenton reaction. We, thus, found a novel phenomenon that free radicals directly affect DJ-1 to form SDS-resistant dimers. Moreover, the formation of the SDS-resistant dimer impaired anti-oxidative stress activity of DJ-1 both in cell viability assay and H₂O₂-elimination assay in vitro. Similar SDS-resistant dimers were steadily formed with several mutants of DJ-1 found in familial PD patients. These findings suggest that DJ-1 is impaired due to the formation of SDS-resistant dimer when the protein is directly attacked by free radicals yielded by external and internal stresses and that the DJ-1 impairment is one of the causes of sporadic PD.     

Structure and host recognition of serotype 13 glycopeptidolipid from Mycobacterium intracellulare

The Mycobacterium avium-M. intracellulare complex (MAIC) is divided into 28 serotypes by a species-specific glycopeptidolipid (GPL). Previously, we clarified the structures of serotype 7 GPL and two methyltransferase genes (orfA and orfB) in serotype 12 GPL. This study elucidated the chemical structure, biosynthesis gene, and host innate immune response of serotype 13 GPL. The oligosaccharide (OSE) structure of serotype 13 GPL was determined to be 4-2'-hydroxypropanoyl-amido-4,6-dideoxy-β-hexose-(1 → 3)-4-O-methyl-α-L-rhamnose-(1 → 3)-α-L-rhamnose-(1 → 3)-α-L-rhamnose-(1 → 2)-α-L-6-deoxy-talose by using chromatography, mass spectrometry, and nuclear magnetic resonance (NMR) analyses. The structure of the serotype 13 GPL was different from those of serotype 7 and 12 GPLs only in O-methylations. We found a relationship between the structure and biosynthesis gene cluster. M. intracellulare serotypes 12 and 13 have a 1.95-kb orfA-orfB gene responsible for 3-O-methylation at the terminal hexose, orfB, and 4-O-methylation at the rhamnose next to the terminal hexose, orfA. The serotype 13 orfB had a nonfunctional one-base missense mutation that modifies serotype 12 GPL to serotype 13 GPL. Moreover, the native serotype 13 GPL was multiacetylated and recognized via Toll-like receptor 2. The findings presented here imply that serotypes 7, 12, and 13 are phylogenetically related and confirm that acetylation of the GPL is necessary for host recognition. This study will promote better understanding of the structure-function relationships of GPLs and may open a new avenue for the prevention of MAIC infections.