Human BIN3 complements the F-actin localization defects caused by loss of Hob3p, the fission yeast homolog of Rvs161p.

The BAR adaptor proteins encoded by the RVS167 and RVS161 genes from Saccharomyces cerevisiae form a complex that regulates actin, endocytosis, and viability following starvation or osmotic stress. In this study, we identified a human homolog of RVS161, termed BIN3 (bridging integrator-3), and a Schizosaccharomyces pombe homolog of RVS161, termed hob3+ (homolog of Bin3). In human tissues, the BIN3 gene was expressed ubiquitously except for brain. S. pombe cells lacking Hob3p were often multinucleate and characterized by increased amounts of calcofluor-stained material and mislocalized F-actin. For example, while wild-type cells localized F-actin to cell ends during interphase, hob3Delta mutants had F-actin patches distributed randomly around the cell. In addition, medial F-actin rings were rarely found in hob3Delta mutants. Notably, in contrast to S. cerevisiae rvs161Delta mutants, hob3Delta mutants showed no measurable defects in endocytosis or response to osmotic stress, yet hob3+ complemented the osmosensitivity of a rvs161Delta mutant. BIN3 failed to rescue the osmosensitivity of rvs161Delta, but the actin localization defects of hob3Delta mutants were completely rescued by BIN3 and partially rescued by RVS161. These findings suggest that hob3+ and BIN3 regulate F-actin localization, like RVS161, but that other roles for this gene have diverged somewhat during evolution.

The thrombospondin motif of aggrecanase-1 (ADAMTS-4) is critical for aggrecan substrate recognition and cleavage.

Aggrecanase-1 (ADAMTS-4) is a member of the a disintegrin and metalloprotease with thrombospondin motifs (ADAMTS) protein family that was recently identified. Aggrecanase-1 is one of two ADAMTS cartilage-degrading enzymes purified from interleukin-1-stimulated bovine nasal cartilage (Tortorella, M. D., Burn, T. C., Pratta, M. A. , Abbaszade, I., Hollis, J. M., Liu, R., Rosenfeld, S. A., Copeland, R. A., Decicco, C. P., Wynn, R., Rockwell, A., Yang, F., Duke, J. L., Solomon, K., George, H., Bruckner, R., Nagase, H., Itoh, Y., Ellis, D. M., Ross, H., Wiswall, B. H., Murphy, K., Hillman, M. C., Jr., Hollis, G. F., and Arner, E.C. (1999) Science 284, 1664-1666; 2 Abbaszade, I., Liu, R. Q., Yang, F., Rosenfeld, S. A., Ross, O. H., Link, J. R., Ellis, D. M., Tortorella, M. D., Pratta, M. A., Hollis, J. M., Wynn, R., Duke, J. L., George, H. J., Hillman, M. C., Jr., Murphy, K., Wiswall, B. H., Copeland, R. A., Decicco, C. P., Bruckner, R., Nagase, H., Itoh, Y., Newton, R. C., Magolda, R. L., Trzaskos, J. M., and Burn, T. C. (1999) J. Biol. Chem. 274, 23443-23450). The aggrecan products generated by this enzyme are found in cartilage cultures stimulated with cytokines and in synovial fluid from patients with arthritis, suggesting that aggrecanase-1 may be important in diseases involving cartilage destruction. Here we demonstrate that the thrombospondin type-1 (TSP-1) motif located within the C terminus of aggrecanase-1 binds to the glycosaminoglycans of aggrecan. Data from several studies indicate that this binding of aggrecanase-1 to aggrecan through the TSP-1 motif is necessary for enzymatic cleavage of aggrecan. 1) A truncated form of aggrecanase-1 lacking the TSP-1 motif was not effective in cleaving aggrecan. 2) Several peptides representing different regions of the TSP-1 motif effectively blocked aggrecanase-1 cleavage of aggrecan by preventing the enzyme from binding to the substrate. 3) Aggrecanase-1 was not effective in cleaving glycosaminoglycan-free aggrecan. Taken together, these data suggest that the TSP-1 motif of aggrecanase-1 is critical for substrate recognition and cleavage.

Sites of aggrecan cleavage by recombinant human aggrecanase-1 (ADAMTS-4).

Aggrecan, the major proteoglycan of cartilage that provides its mechanical properties of compressibility and elasticity, is one of the first matrix components to undergo measurable loss in arthritic diseases. Two major sites of proteolytic cleavage have been identified within the interglobular domain (IGD) of the aggrecan core protein, one between amino acids Asn(341)-Phe(342) which is cleaved by matrix metalloproteinases and the other between Glu(373)-Ala(374) that is attributed to aggrecanase. Although several potential aggrecanase-sensitive sites had been identified within the COOH terminus of aggrecan, demonstration that aggrecanase cleaved at these sites awaited isolation and purification of this protease. We have recently cloned human aggrecanase-1 (ADAMTS-4) (Tortorella, M. D., Burn, T. C., Pratta, M. A., Abbaszade, I., Hollis, J. M., Liu, R., Rosenfeld, S. A., Copeland, R. A., Decicco, C. P., Wynn, R., Rockwell, A., Yang, F., Duke, J. L., Solomon, K., George, H., Bruckner, R., Nagase, H., Itoh, Y., Ellis, D. M., Ross, H., Wiswall, B. H., Murphy, K., Hillman, M. C., Jr., Hollis, G. F., Newton, R. C., Magolda, R. L., Trzaskos, J. M., and Arner, E. C. (1999) Science 284, 1664-1666) and herein demonstrate that in addition to cleavage at the Glu(373)-Ala(374) bond, this protease cleaves at four sites within the chondroitin-sulfate rich region of the aggrecan core protein, between G2 and G3 globular domains. Importantly, we show that this cleavage occurs more efficiently than cleavage within the IGD at the Glu(373)-Ala(374) bond. Cleavage occurred preferentially at the KEEE(1667-1668)GLGS bond to produce both a 140-kDa COOH-terminal fragment and a 375-kDa fragment that retains an intact G1. Cleavage also occurred at the GELE(1480-1481)GRGT bond to produce a 55-kDa COOH-terminal fragment and a G1-containing fragment of 320 kDa. Cleavage of this 320-kDa fragment within the IGD at the Glu(373)-Ala(374) bond then occurred to release the 250-kDa BC-3-reactive fragment from the G1 domain. The 140-kDa GLGS-reactive fragment resulting from the preferential cleavage was further processed at two additional cleavage sites, at TAQE(1771)-(1772)AGEG and at VSQE(1871-1872)LGQR resulting in the formation of a 98-kDa fragment with an intact G3 domain and two small fragments of approximately 20 kDa. These data elucidate the sites and efficiency of cleavage during aggrecan degradation by aggrecanase and suggest potential tools for monitoring aggrecan cleavage in arthritis.

Possible role of valvular serotonin 5-HT(2B) receptors in the cardiopathy associated with fenfluramine.

Dexfenfluramine was approved in the United States for long-term use as an appetite suppressant until it was reported to be associated with valvular heart disease. The valvular changes (myofibroblast proliferation) are histopathologically indistinguishable from those observed in carcinoid disease or after long-term exposure to 5-hydroxytryptamine (5-HT)(2)-preferring ergot drugs (ergotamine, methysergide). 5-HT(2) receptor stimulation is known to cause fibroblast mitogenesis, which could contribute to this lesion. To elucidate the mechanism of "fen-phen"-associated valvular lesions, we examined the interaction of fenfluramine and its metabolite norfenfluramine with 5-HT(2) receptor subtypes and examined the expression of these receptors in human and porcine heart valves. Fenfluramine binds weakly to 5-HT(2A), 5-HT(2B), and 5-HT(2C) receptors. In contrast, norfenfluramine exhibited high affinity for 5-HT(2B) and 5-HT(2C) receptors and more moderate affinity for 5-HT(2A) receptors. In cells expressing recombinant 5-HT(2B) receptors, norfenfluramine potently stimulated the hydrolysis of inositol phosphates, increased intracellular Ca(2+), and activated the mitogen-activated protein kinase cascade, the latter of which has been linked to mitogenic actions of the 5-HT(2B) receptor. The level of 5-HT(2B) and 5-HT(2A) receptor transcripts in heart valves was at least 300-fold higher than the levels of 5-HT(2C) receptor transcript, which were barely detectable. We propose that preferential stimulation of valvular 5-HT(2B) receptors by norfenfluramine, ergot drugs, or 5-HT released from carcinoid tumors (with or without accompanying 5-HT(2A) receptor activation) may contribute to valvular fibroplasia in humans.

Cloning and characterization of ADAMTS11, an aggrecanase from the ADAMTS family.

Aggrecan is responsible for the mechanical properties of cartilage. One of the earliest changes observed in arthritis is the depletion of cartilage aggrecan due to increased proteolytic cleavage within the interglobular domain. Two major sites of cleavage have been identified in this region at Asn(341)-Phe(342) and Glu(373)-Ala(374). While several matrix metalloproteinases have been shown to cleave at Asn(341)-Phe(342), an as yet unidentified protein termed "aggrecanase" is responsible for cleavage at Glu(373)-Ala(374) and is hypothesized to play a pivotal role in cartilage damage. We have identified and cloned a novel disintegrin metalloproteinase with thrombospondin motifs that possesses aggrecanase activity, ADAMTS11 (aggrecanase-2), which has extensive homology to ADAMTS4 (aggrecanase-1) and the inflammation-associated gene ADAMTS1. ADAMTS11 possesses a number of conserved domains that have been shown to play a role in integrin binding, cell-cell interactions, and extracellular matrix binding. We have expressed recombinant human ADAMTS11 in insect cells and shown that it cleaves aggrecan at the Glu(373)-Ala(374) site, with the cleavage pattern and inhibitor profile being indistinguishable from that observed with native aggrecanase. A comparison of the structure and expression patterns of ADAMTS11, ADAMTS4, and ADAMTS1 is also described. Our findings will facilitate the study of the mechanisms of cartilage degradation and provide targets to search for effective inhibitors of cartilage depletion in arthritic disease.

Purification and cloning of aggrecanase-1: a member of the ADAMTS family of proteins.

We purified, cloned, and expressed aggrecanase, a protease that is thought to be responsible for the degradation of cartilage aggrecan in arthritic diseases. Aggrecanase-1 [a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4)] is a member of the ADAMTS protein family that cleaves aggrecan at the glutamic acid-373-alanine-374 bond. The identification of this protease provides a specific target for the development of therapeutics to prevent cartilage degradation in arthritis.

Isolation of a new mouse 3beta-hydroxysteroid dehydrogenase isoform, 3beta-HSD VI, expressed during early pregnancy.

The enzyme 3beta-hydroxysteroid dehydrogenase (3beta-HSD) is a key enzyme in the biosynthesis of steroid hormones. To date, this laboratory has isolated and characterized five distinct 3beta-HSD complementary DNAs (cDNAs) in the mouse (3beta-HSD I through V). These different forms are expressed in a tissue- and developmentally-specific manner and fall into two functionally distinct enzymes. 3beta-HSD I and III, and most likely II, function as dehydrogenase/isomerases, whereas 3beta-HSD IV and V function as 3-ketosteroid reductases. This study describes the isolation, characterization, and tissue-specific expression of a sixth member of this gene family, 3beta-HSD VI. This new isoform functions as an NAD+-dependent dehydrogenase/isomerase exhibiting very low Michaelis-Menten constant (Km) values for pregnenolone (approximately 0.035 microM) and dehydroepiandrosterone (approximately 0.12 microM). 3beta-HSD VI is the earliest isoform to be expressed during embryogenesis in cells of embryonic origin at 7 and 9.5 days postcoitum (pc), and is the major isoform expressed in uterine tissue at the time of implantation (4.5 days pc) and continues to be expressed in uterine tissue at 6.5, 7.5, and 9.5 days pc. 3beta-HSD VI is expressed in giant trophoblasts at 9.5 days pc and is expressed in the placenta through day 15.5 pc. In the adult mouse, 3beta-HSD VI appears to be the only isoform expressed in the skin and also is expressed in the testis, but to a lesser extent than 3beta-HSD I. Mouse 3beta-HSD VI cDNA is orthologous to human 3beta-HSD I cDNA. Human type I 3beta-HSD has been shown to be the only isoform expressed in the placenta and skin. The demonstration that mouse 3beta-HSD VI functions as a dehydrogenase/isomerase and is the predominant isoform expressed during the first half of pregnancy in uterine tissue and in embryonic cells suggests that this isoform may be involved in local production of progesterone, which is needed for successful implantation of the blastocyst and/or maintenance of early pregnancy.

The multiple murine 3 beta-hydroxysteroid dehydrogenase isoforms: structure, function, and tissue- and developmentally specific expression.

The enzyme 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) is essential for the biosynthesis of all active steroid hormones. To date five distinct isoforms have been identified in the mouse. The different isoforms are indicated by roman numerals (I-V) in the chronological order in which they have been isolated. The different isoforms are expressed in a tissue- and developmentally specific manner and fall into two functionally distinct groups. 3 beta-HSD I, II, and III function as NAD(+)-dependent dehydrogenaselisomerases, and IV and V function as NADPH-dependent 3-keto steroid reductases. These latter two isoforms, therefore, are not involved in the biosynthesis of steroid hormones, but most likely in the inactivation of steroid hormones. In the adult mouse 3 beta-HSD I is expressed in the classical steroidogenic tissues, the adrenal glands and the gonads. 3 beta-HSD II and III are expressed in the liver and kidney, with III being the major isoform expressed in the adult liver. 3 beta-HSD IV is expressed almost exclusively in the kidney of both sexes, and expression of 3 beta-HSD V is observed only in the male liver starting late in puberty. In the fetal liver of both sexes, 3 beta-HSD I is the major or only isoform expressed at 13.5 days postconception and remains the major isoform until the day of birth, after which 3 beta-HSD III becomes the major isoform. Expression of 3 beta-HSD I in the liver decreases after birth and ceases by day 20 postnatally. Thus the liver expresses four distinct isoforms of 3 beta-HSD, I, II, III, and V, at different times during development. The mouse 3 beta-HSD genes, Hsd3b, have been mapped to a small region on mouse chromosome 3. Analysis of two yeast artificial chromosome (YAC) libraries identified one clone that contains the entire Hsd3b locus within a 1400-kb insert. Hybridization by Southern blot analysis of restriction-enzyme-digested YAC DNA using an 18-base oligonucleotide that hybridizes without mismatch to all known Hsd3b sequences indicates that there are a total of seven Hsd3b genes or pseudogenes in the mouse genome. Further analysis of mouse genomic DNA by pulse field gel electrophoresis suggests that all of the Hsd3b gene family is found within a 400-kb fragment.

Expression of multiple forms of 3 beta-hydroxysteroid dehydrogenase in the mouse liver during fetal and postnatal development.

The enzyme 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) is essential for the biosynthesis of all steroid hormones. To date this laboratory has isolated and characterized five distinct 3 beta HSD cDNAs in the mouse (3 beta HSD I-V). The different isoforms fall into two functionally distinct groups. 3 beta HSD I and III function as dehydrogenase/isomerases and 3 beta HSD IV and V function as 3-ketosteroid reductases. Previously it was shown that the liver of the adult mouse expresses 3 beta HSD II, III and V, with 3 beta HSD III being the major isoform. This study examines the expression of the different forms of 3 beta HSD mRNAs and proteins in the livers of male and female mice during fetal and postnatal development. 3 beta HSD I, which in the adult mouse is expressed only in the gonads and adrenal glands, is the major isoform expressed in both male and female livers during fetal development until the first postnatal (pn) day after which time 3 beta HSD III becomes the major isoform. Expression of 3 beta HSD I mRNA and protein completely ceases after day 20 pn. The expression of 3 beta HSD V is first detected at day 40 pn and is observed only in the male. Very low expression of 3 beta HSD II mRNA is detected throughout development. Previous characterization of enzymatic activity of the expressed proteins showed that 3 beta HSD I exhibits lower Km values for the delta 5-3 beta-hydroxysteroids than 3 beta HSD III, indicating that 3 beta HSD I functions as a more efficient 3 beta-hydroxysteroid dehydrogenase/isomerase than 3 beta HSD III. The results of this study suggest that the liver may play an important role in the biosynthesis of steroid hormones during murine fetal development.

The mouse 3 beta-hydroxysteroid dehydrogenase multigene family includes two functionally distinct groups of proteins.

The enzyme 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) plays an essential role in the biosynthesis of all steroid hormones. We previously reported the isolation, characterization, and tissue-specific expression of four distinct but highly homologous 3 beta HSD cDNAs (forms I, II, III, and IV). Enzymatic characterization of three of these isoforms demonstrated that mouse 3 beta HSD I and III function as dehydrogenase/isomerases, but 3 beta HSD IV functions exclusively as a 3-ketosteroid reductase. We now report the isolation and characterization of an additional distinct mouse 3 beta HSD cDNA, 3 beta HSD V, which is expressed in the liver of male mice beginning in late puberty. Similar to 3 beta HSD IV, 3 beta HSD V functions exclusively as a 3-ketosteroid reductase converting an active androgen, dihydrotestosterone (DHT), into an inactive androgen, 5 alpha-androstane-3 beta,17 beta-diol. Expressed 3 beta HSD V, however, exhibits a considerably lower apparent Michaelis-Menten constant (Km) value for DHT than 3 beta HSD IV (0.47 microM vs. 2.2 microM, respectively). The complete predicted amino acid sequence of 3 beta HSD II is also reported. The predicted amino acid sequence of mouse 3 beta HSD V reveals that this new form is more closely related to the 3-ketosteroid reductases, mouse 3 beta HSD IV and rat III (93 and 84% identity, respectively), than to the other rodent isoforms that share less than 75% identity.(ABSTRACT TRUNCATED AT 250 WORDS)