An interaction between frataxin and Isu1/Nfs1 that is crucial for Fe/S cluster synthesis on Isu1

Depletion of the mitochondrial matrix protein frataxin is the molecular cause of the neurodegenerative disease Friedreich ataxia. The function of frataxin is unclear, although recent studies have suggested a function of frataxin (yeast Yfh1) in iron/sulphur (Fe/S) protein biogenesis. Here, we show that Yfh1 specifically binds to the central Fe/S-cluster (ISC)-assembly complex, which is composed of the scaffold protein Isu1 and the cysteine desulphurase Nfs1. Association between Yfh1 and Isu1/Nfs1 was markedly increased by ferrous iron, but did not depend on ISCs on Isu1. Functional analyses in vivo showed an involvement of Yfh1 in de novo ISC synthesis on Isu1. Our data demonstrate a crucial function of Yfh1 in Fe/S protein biogenesis by defining its function in an early step of this essential process. The iron-dependent binding of Yfh1 to Isu1/Nfs1 suggests a role of frataxin/Yfh1 in iron loading of the Isu scaffold proteins.     

The biology of VEGF and its receptors

Vascular endothelial growth factor (VEGF) is a key regulator of physiological angiogenesis during embryogenesis, skeletal growth and reproductive functions. VEGF has also been implicated in pathological angiogenesis associated with tumors, intraocular neovascular disorders and other conditions. The biological effects of VEGF are mediated by two receptor tyrosine kinases (RTKs), VEGFR-1 and VEGFR-2, which differ considerably in signaling properties. Non-signaling co-receptors also modulate VEGF RTK signaling. Currently, several VEGF inhibitors are undergoing clinical testing in several malignancies. VEGF inhibition is also being tested as a strategy for the prevention of angiogenesis, vascular leakage and visual loss in age-related macular degeneration.     

Components involved in assembly and dislocation of iron-sulfur clusters on the scaffold protein Isu1p

The mitochondrial proteins Isu1p and Isu2p play an essential role in the maturation of cellular iron-sulfur (Fe/S) proteins in eukaryotes. By radiolabelling of yeast cells with 55Fe we demonstrate that Isu1p binds an oxygen-resistant non-chelatable Fe/S cluster providing in vivo evidence for a scaffolding function of Isu1p during Fe/S cluster assembly. Depletion of the cysteine desulfurase Nfs1p, the ferredoxin Yah1p or the yeast frataxin homologue Yfh1p by regulated gene expression causes a strong decrease in the de novo synthesis of Fe/S clusters on Isu1p. In contrast, depletion of the Hsp70 chaperone Ssq1p, its co-chaperone Jac1p or the glutaredoxin Grx5p markedly increased the amount of Fe/S clusters bound to Isu1p, even though these mitochondrial proteins are crucial for maturation of Fe/S proteins. Hence Ssq1p/Jac1p and Grx5p are required in a step after Fe/S cluster synthesis on Isu1p, for instance in dissociation of preassembled Fe/S clusters from Isu1p and/or their insertion into apoproteins. We propose a model that dissects Fe/S cluster biogenesis into two major steps and assigns its central components to one of these two steps.     

Bone morphogenetic protein-7 (OP1) and transforming growth factor-beta1 modulate 1,25(OH)2-vitamin D3-induced differentiation of human osteoblasts

Bone morphogenetic proteins (BMPs) and transforming growth factor-beta (TGFbeta) are potent regulators of osteoblast differentiation and proliferation, processes that are crucial in bone remodeling. BMPs and TGFbeta act in concert with other local factors and hormones, among them 1,25(OH)2-vitamin D3 and insulin. Here we show that BMP7 inhibits 1,25(OH)2-vitamin D3-induced differentiation of human osteoblasts, whereas TGFbeta1 stimulates it, as assessed by assays for alkaline phosphatase (ALP) induction, matrix mineralization, and morphology changes. BMP7 or TGFbeta1 alone affects the differentiation of human osteoblasts. Similar results were obtained in assays for ALP induction using conditionally immortalized human osteoblasts (hFOB) and primary osteoblasts obtained from trabecular bone of the femoral head after hip replacement surgery. BMP7 stimulation led to a decrease of 1,25(OH)2-vitamin D3-induced binding of nuclear proteins to a vitamin D response element, as shown by electrophoretic mobility shift assay. Our results suggest that 1,25(OH)2-vitamin D3 modulates in opposite ways the effects of BMP7 and TGFbeta1 on osteoblast differentiation.     

Localization of HCMV UL33 and US27 in endocytic compartments and viral membranes

The human cytomegalovirus genome encodes four putative seven transmembrane domain chemokine receptor-like proteins. Although important in viral pathogenesis, little is known about the properties or functions of these proteins. We previously reported that US28 is located in endocytic vesicles and undergoes constitutive endocytosis and recycling. Here we studied the cellular distributions and trafficking of two other human cytomegalovirus chemokine receptor-like proteins, UL33 and US27, in transfected and human cytomegalovirus-infected cells. Immunofluorescence staining indicated that UL33 and US27 are located at the cell surface, although the majority of both proteins was seen in intracellular organelles located in the perinuclear region of the cell. The intracellular pools of UL33 and US27 showed overlap with markers for endocytic organelles. Antibody-feeding experiments indicated that cell surface US27 undergoes endocytosis. By immunogold labeling of cryosections and electron microscopy, UL33 was seen to localize to multivesicular bodies (MVBs or multivesicular endosomes). Electron microscopy analysis of human cytomegalovirus-infected cells showed that most virus particles wrapped individually into short membrane cisternae, although virus particles were also occasionally seen within and budding into MVBs. Electron microscopy immunolocalization of viral UL33 and US27 on ultrathin cryosections of human cytomegalovirus-infected cells showed gold particles over the membranes into which virions were wrapping, in small membrane tubules and vesicles and in MVBs. Labeling of the human cytomegalovirus glycoproteins gB and gH indicated that these proteins were also present in the same membrane structures. This first electron microscopy analysis of human cytomegalovirus assembly using immunolabeling suggests that the localization of UL33, US27 and US28 to endosomes may allow these proteins to be incorporated into the viral membrane during the final stages of human cytomegalovirus assembly.     

Dual role of the RIC-3 protein in trafficking of serotonin and nicotinic acetylcholine receptors

The ric-3 gene is required for maturation of nicotinic acetylcholine receptors in Caenorhabditis elegans. The human homolog of RIC-3, hRIC-3, enhances expression of alpha7 nicotinic receptors in Xenopus laevis oocytes, whereas it totally abolishes expression of alpha4beta2 nicotinic and 5-HT3 serotonergic receptors. Both the N-terminal region of hRIC-3, which contains two transmembrane segments, and the C-terminal region are needed for these differential effects. hRIC-3 inhibits receptor expression by hindering export of mature receptors to the cell membrane. By using chimeric proteins made of alpha7 and 5-HT3 receptors, we have shown that the presence of an extracellular isoleucine close to the first transmembrane receptor fragment is responsible for the transport arrest induced by hRIC-3. Enhancement of alpha7 receptor expression occurs, at least, at two levels: by increasing the number of mature receptors and facilitating its transport to the membrane. Certain amino acids of a putative amphipathic helix present at the large cytoplasmic region of the alpha7 subunit are required for these actions. Therefore, hRIC-3 can act as a specific regulator of receptor expression at different levels.     

The identification of a minimal dimerization motif QXXS that enables homo- and hetero-association of transmembrane helices in vivo

Assembly of transmembrane (TM) domains is a critical step in the function of membrane proteins, and therefore, determining the amino acid motifs that mediate this process is important. Studies along this line have shown that the GXXXG motif is involved in TM assembly. In this study we characterized the minimal dimerization motif in the bacterial Tar-1 homodimer TM domain, which does not contain a GXXXG sequence. We found that a short polar motif QXXS is sufficient to induce stable TM-TM interactions. Statistical analysis revealed that this motif appears to be significantly over-represented in a bacterial TM data base compared with its theoretical expectancy, suggesting a general role for this motif in TM assembly. A truncated short TM peptide (9 residues) that contains the QXXS motif interacted slightly with the wild-type Tar-1. However, the same short TM peptide regained wild-type-like activity when conjugated to an octanoyl aliphatic moiety. Biophysical studies indicated that this modification compensated for the missing hydrophobicity, stabilized alpha-helical structure, and enabled insertion of the peptide into the membrane core. These findings serve as direct evidence that even a short peptide containing a minimal recognition motif is sufficient to inhibit the proper assembly of TM domains. Interestingly, electron microscopy revealed that above the critical micellar concentration, the TM lipopeptide forms a network of nanofibers, which can serve for the slow release of the active lipopeptide.     

Urinary Progestogen and Estrogen Excretion During Pregnancy in Eulemur macaco flavifrons,E. rubriventer,and Hapalemur griseus occidentalis

Via a non-invasive approach, we aimed to provide comparative data on the presence and relative abundance of progestogen and estrogen metabolites excreted into the urine of Eulemur rubriventer, E. macaco flavifrons and Hapalemur griseus occidentalis and to characterize the patterns of progestogen and estrogen excretion during pregnancy. We found that estrone is the major urinary estrogen in Eulemur macaco flavifrons and Hapalemur griseus occidentalis, while 16-hydroxyestrone appeared to be the predominant estrogen in the urine of E. rubriventer. Estradiol-17ß was either absent (Hapalemur) or present in very low amounts. HPLC of progestogens had high levels of immunoreactivity in an assay against 5-pregnane-3-ol-20-one (5-P-3OH) in each species, though the nature of the progestogens measured by the 5-P-3OH assay differed among species. During pregnancy, 5-P-3OH levels remained relatively low in Eulemur rubriventer, whereas in E. macaco flavifrons and Hapalemur griseus occidentalis, a sustained elevation in levels occurred from mid-pregnancy until the final weeks before parturition. Estrogen excretion differed depending on the sex of the fetus in each species, with only females carrying male infants showing clearly elevated estrogen levels during the last 6–8 weeks of gestation. In conclusion, we demonstrate both species similarities and differences in the metabolism and urinary excretion patterns of reproductive hormones throughout gestation in the Lemuridae. The data not only extend our knowledge on the reproductive physiology of lemurs but also show that more studies on other lemur taxa are needed to provide a broader basis for interspecific comparison.     

VEGF-null cells require PDGFR alpha signaling-mediated stromal fibroblast recruitment for tumorigenesis

We generated VEGF-null fibrosarcomas from VEGF-loxP mouse embryonic fibroblasts to investigate the mechanisms of tumor escape after VEGF inactivation. These cells were found to be tumorigenic and angiogenic in vivo in spite of the absence of tumor-derived VEGF. However, VEGF derived from host stroma was readily detected in the tumor mass and treatment with a newly developed anti-VEGF monoclonal antibody substantially inhibited tumor growth. The functional significance of stroma-derived VEGF indicates that the recruitment of stromal cells is critical for the angiogenic and tumorigenic properties of these cells. Here we identified PDGF AA as the major stromal fibroblast chemotactic factor produced by tumor cells, and demonstrated that disrupting the paracrine PDGFR alpha signaling between tumor cells and stromal fibroblasts by soluble PDGFR alpha-IgG significantly reduced tumor growth. Thus, PDGFR alpha signaling is required for the recruitment of VEGF-producing stromal fibroblasts for tumor angiogenesis and growth. Our findings highlight a novel aspect of PDGFR alpha signaling in tumorigenesis.