BBA - General Subjects (v.1800, #4)

Effect of fumagillin on adipocyte differentiation and adipogenesis by I. Scroyen; V. Christiaens; H.R. Lijnen (425-429).
Inhibition of angiogenesis may impair adipose tissue development.The effect of fumagillin (a methionine aminopeptidase-2 inhibitor) on adipocyte differentiation and de novo adipogenesis was investigated in murine model systems.During in vitro differentiation of murine 3T3-F442A preadipocytes, administration of fumagillin (≥ 1 μM) resulted in reduced expression of methionine aminopeptidase-2, and in enhanced differentiation rate. In vivo, de novo development of adipose tissue following injection of preadipocytes in nude mice kept on high fat diet was somewhat, but not significantly (p  = 0.06), reduced by administration of fumagillin (1 mg/kg/day during 4 weeks by oral gavage). This was not associated with effects on blood vessel size or density, whereas blood vessel density normalized to adipocyte density was enhanced upon fumagillin treatment. In vivo BrdU incorporation experiments did not reveal effects of fumagillin on cell proliferation in adipose tissues, and cellular apoptosis was also not affected.Treatment with fumagillin enhances in vitro differentiation of preadipocytes, but has only a minor effect on in vivo adipogenesis.These studies on in vitro and in vivo preadipcoyte differentiation thus do not support an anti-obesity effect of fumagillin as a result of effects on adipocyte differentiation.
Keywords: Adipose tissue; Angiogenesis; Fumagillin; Adipogenesis; Adipocyte;

Cloning and molecular characterization of Dashurin encoded by C20orf116, a PCI-domain containing protein by D. Neziri; A. Ilhan; M. Maj; O. Majdic; S. Baumgartner-Parzer; G. Cohen; W. Base; L. Wagner (430-438).
Characterization of gene products originating from undefined open reading frames and delineation of biological functions has become the task after the human genome has been decoded.We cloned the human C20orf 116 and defined its transcript in liver, kidney and various brain regions by Northern analysis. Antibodies against recombinant protein used for immunofluorescence and immunoblots confirmed its expression in these tissues. With the focus on kidney, its tubular expression and presence in glomerula were shown.A 28 aa long signal peptide predicted by in silico analysis is reflected by visualization of size variants of ∼ 3 kDa difference suggesting a signal peptidase cleavage of the proform. Cell compartment separation confirmed the presence of Dashurin in peroxisomes/mitochondria, microsomes, cytosol and nucleus. This is in line with green fluorescent protein (GFP)-Dashurin fusion protein shuttling between cytosol and nucleus. Luciferase reporter studies revealed a 2–3 fold increase of promoter activities upon over-expression. Bioinformatic analysis identified a PCI-domain at the C-terminus providing protein–protein interaction capabilities.Our present findings suggest the involvement of Dashurin in gene transcription or mRNA translation.Dashurin shares the PCI-domain with three multisubunit protein complexes (26S proteasome, COP9 signalosome and eIF3 translation initiation factor).
Keywords: C20orf116; PCI-domain; GFP; Transcription; Translation; Dashurin;

Plagiochin E (PLE) is an antifungal active macrocyclic bis(bibenzyl) isolated from liverwort Marchantia polymorpha L. To elucidate the mechanism of action, previous studies revealed that the antifungal effect of PLE was associated with the accumulation of ROS, an important regulator of apoptosis in Candida albicans. The present study was designed to find whether PLE caused apoptosis in C. albicans.We assayed the cell cycle by flow cytometry using PI staining, observed the ultrastructure by transmission electron microscopy, studied the nuclear fragmentation by DAPI staining, and investigated the exposure of phosphatidylserine at the outer layer of the cytoplasmic membrane by the FITC-annexin V staining. The effect of PLE on expression of CDC28, CLB2, and CLB4 was determined by RT-PCR. Besides, the activity of metacaspase was detected by FITC-VAD-FMK staining, and the release of cytochrome c from mitochondria was also determined. Furthermore, the effect of antioxidant L-cysteine on PLE-induced apoptosis in C. albicans was also investigated.Cells treated with PLE showed typical markers of apoptosis: G2/M cell cycle arrest, chromatin condensation, nuclear fragmentation, and phosphatidylserine exposure. The expression of CDC28, CLB2, and CLB4 was down-regulated by PLE, which may contribute to PLE-induced G2/M cell cycle arrest. Besides, PLE promoted the cytochrome c release and activated the metacaspase, which resulted in the yeast apoptosis. The addition of L-cysteine prevented PLE-induced nuclear fragmentation, phosphatidylserine exposure, and metacaspase activation, indicating the ROS was an important mediator of PLE-induced apoptosis.PLE induced apoptosis in C. albicans through a metacaspase-dependent apoptotic pathway.In this study, we reported for the first time that PLE induced apoptosis in C. albicans through activating the metacaspase. These results would conduce to elucidate its underlying antifungal mechanism.
Keywords: Apoptosis; Candida albicans; Cell cycle arrest; Metacaspase; Plagiochin E;

Increased plasticity of the nuclear envelope and hypermobility of telomeres due to the loss of A–type lamins by Winnok H. De Vos; Frederik Houben; Ron A. Hoebe; Raoul Hennekam; Baziel van Engelen; Erik M.M. Manders; Frans C.S. Ramaekers; Jos L.V. Broers; Patrick Van Oostveldt (448-458).
The nuclear lamina provides structural support to the nucleus and has a central role in defining nuclear organization. Defects in its filamentous constituents, the lamins, lead to a class of diseases collectively referred to as laminopathies. On the cellular level, lamin mutations affect the physical integrity of nuclei and nucleo-cytoskeletal interactions, resulting in increased susceptibility to mechanical stress and altered gene expression.In this study we quantitatively compared nuclear deformation and chromatin mobility in fibroblasts from a homozygous nonsense LMNA mutation patient and a Hutchinson–Gilford progeria syndrome patient with wild type dermal fibroblasts, based on the visualization of mCitrine labeled telomere-binding protein TRF2 with light-economical imaging techniques and cytometric analyses.Without application of external forces, we found that the absence of functional lamin A/C leads to increased nuclear plasticity on the hour and minute time scale but also to increased intranuclear mobility down to the second time scale. In contrast, progeria cells show overall reduced nuclear dynamics. Experimental manipulation (farnesyltransferase inhibition or lamin A/C silencing) confirmed that these changes in mobility are caused by abnormal or reduced lamin A/C expression.These observations demonstrate that A-type lamins affect both nuclear membrane and telomere dynamics.Because of the pivotal role of dynamics in nuclear function, these differences likely contribute to or represent novel mechanisms in laminopathy development.
Keywords: Lamin A/C; LMNA; Telomere; TRF2; CLEM; HGPS; Nuclear dynamics;

Enhanced thermal and ultrasonic stability of a fungal protease encapsulated within biomimetically generated silicate nanospheres by Ashkan Madadlou; Daniela Iacopino; David Sheehan; Zahra Emam-Djomeh; Mohammad E. Mousavi (459-465).
Dendrimers are highly branched synthetic macromolecules with a globular shape. They have been successfully used for generation of nanospheres at mild conditions via biomimetic silicification. Encapsulation of enzyme molecules within these nanospheres during their synthesis is a promising method for rapid and efficient entrapment of several enzymes. However, encapsulation of proteolytic enzymes has been rarely done via biomimetic silicification. As well, the operational stability of encapsulated enzyme has not been systematically reported.A proteolytic enzyme, either α-Chymotrypsin or a fungal protease from Aspergilus Oryzea was encapsulated along with iron oxide nanoparticles within particles yielded via biomimetic silicification of different generations of polyamidoamine (PAMAM) dendrimers. Stability of encapsulated enzyme was compared to that of free enzyme during storage at room temperature. As well, their thermal and ultrasonic stabilities were measured. Scanning electron microscopy, transmission electron microscopy and optical microscopy were used to investigate the morphology of nanospheres.Determination of encapsulation efficiency revealed that ∼ 85% of fungal protease with concentration 1.4 mg mL− 1 stock solution was immobilized within particles yielded by generation 0. Based on microscopic images the generated particles interconnected with each other and had spherical morphologies independent of generation. Kinetic analysis of encapsulated fungal protease demonstrated that Mechaelis-Menten constant (K m ) slightly increased.PAMAM dendrimer generation 0 could be effectively used for rapid encapsulation of a fungal protease from Aspegilus Oryzae. Encapsulation significantly enhances the thermal and ultrasonic stabilities of enzymes, suggesting a range of diverse applications for them.
Keywords: Enzyme; Dendrimer; Immobilization; Microscopy; Biomimetic silicification; Ultrasound;

The Theta class glutathione transferase GST T1-1 is a ubiquitously occurring detoxication enzyme. The rat and mouse enzymes have high catalytic activities with numerous electrophilic compounds, but the homologous human GST T1-1 has comparatively low activity with the same substrates. A major structural determinant of substrate recognition is the H-site, which binds the electrophile in proximity to the nucleophilic sulfur of the second substrate glutathione. The H-site is formed by several segments of amino acid residues located in separate regions of the primary structure. The C-terminal helix of the protein serves as a lid over the active site, and contributes several residues to the H-site.Site-directed mutagenesis of the H-site in GST T1-1 was used to create the mouse Arg234Trp for comparison with the human Trp234Arg mutant and the wild-type rat, mouse, and human enzymes. The kinetic properties were investigated with an array of alternative electrophilic substrates to establish substrate selectivity profiles for the different GST T1-1 variants.The characteristic activity profile of the rat and mouse enzymes is dependent on Arg in position 234, whereas the human enzyme features Trp. Reciprocal mutations of residue 234 between the rodent and human enzymes transform the substrate-selectivity profiles from one to the other.H-site residue 234 has a key role in governing the activity and substrate selectivity profile of GST T1-1.The functional divergence between human and rodent Theta class GST demonstrates that a single point mutation can enable or suppress enzyme activities with different substrates.
Keywords: Glutathione transferase; Active-site residue; Alternative substrate; Detoxication; Haloalkane;

A nuclear ligand MRG15 involved in the proapoptotic activity of medicinal fungal galectin AAL (Agrocybe aegerita lectin) by Yi Liang; Jia Cheng Lin; Kun Wang; Yi Jie Chen; Hong Hong Liu; Rong Luan; Shuai Jiang; Tao Che; Yong Zhao; De Feng Li; Da Cheng Wang; Lin Guo; Hui Sun (474-480).
We have previously reported a novel fungal galectin Agrocybe aegerita lectin (AAL) with apoptosis-induced activity and nuclear migration activity. The importance of nuclear localization for AAL's apoptosis-induced activity has been established by mutant study. However, the mechanism remains unclear.We further investigated the mechanism using a previously reported carbohydrate recognition domain (CRD) mutant protein H59Q, which retained its nuclear localization activity but lost most of its apoptotic activity. The cell membrane-binding ability of recombinant AAL (rAAL) and H59Q was analyzed by FACS, and their cellular partners were identified by affinity chromatography and mass spectroscopy. Furthermore, the interaction of AAL and ligand was proved by mammalian two-hybrid and pull down assays. A knockdown assay was used to confirm the role of the ligand.The apoptotic activity of AAL could be blocked by lactose. Mutant H59Q retained comparable cell membrane-binding ability to rAAL. Four cellular binding partners of AAL in HeLa cells were identified: glucose-regulated protein 78 (GRP78); mortality factor 4-like protein 1 (MRG15); elongation factor 2 (EEF2); and heat shock protein 70 (Hsp70). CRD region of AAL was required for the interaction between AAL/mutant AAL and MRG15. MRG15 knockdown increased the cells' resistance to AAL treatment.MRG15 was a nuclear ligand for AAL in HeLa cells. These data implied the existence of a novel nuclear pathway for the antitumor activity of fungal galectin AAL.These findings provide a novel explanation of AAL bioactivity and contribute to the understanding of mushroom lectins' antitumor activity.
Keywords: Medicinal mushroom; Lectin; CRD; Affinity chromatography; Ligand;

Human CRISP-3 binds serum α1B-glycoprotein across species by Lene Udby; Anders H. Johnsen; Niels Borregaard (481-485).
CRISP-3 was previously shown to be bound to α1B-glycoprotein (A1BG) in human serum/plasma. All mammalian sera are supposed to contain A1BG, although its presence in rodent sera is not well-documented. Since animal sera are often used to supplement buffers in experiments, in particular such that involve cell cultures, binding proteins present in sera might interfere in the experiments.We examined sera from five different animal species for CRISP-3 binding proteins using gel filtration and ligand blotting. We developed a rapid method for isolation of proteins that bind to human CRISP-3 and identified the isolated proteins by mass spectrometry and N-terminal sequencing.We identified A1BG as a CRISP-3 binding protein in sera from cow, horse and rabbit. CRISP-3 bound kininogen 1 in mouse serum, whereas rat serum showed no CRISP-3 binding activity. In equine serum, we furthermore detected a possible CRISP, already bound to A1BG.It seems to be a common mechanism that A1BGs bind CRISPs, also across species. Apart from the possible physiological implications hereof, complex binding of CRISPs by A1BG (and other proteins) may interfere with the detection and function of CRISPs, when these are studied in the presence of animal sera.
Keywords: Cysteine-rich secretory protein; α1B-glycoprotein; Kininogen; Animal serum; Protein complex; Mass spectrometry;