The subtilisin-like prohormone convertase PC2 and the polypeptide 7B2 (an intracellularly cleaved protein of unknown function) are both selectively present in the regulated secretory pathway of neurons and endocrine cells.Here we demonstrate that intact recombinant 7B2 is a potent inhibitor of PC2 and prevents proPC2 cleavage in vitro, whereas the 7B2 cleavage product is virtually inactive. The PC2-related proteinase PC1/PC3 is not inhibited by 7B2.Furthermore, the carboxyl-terminal half of the 7B2 protein sequence is distantly related to the so-called potato inhibitor I family (which includes subtilisin inhibitors Synthetic compounds (e.g., peptidyl chloromethanes containing basic amino acid residues) have been shown to inhibit the Kex2 enzyme and furin-mediated cleavage of the human immunodeficiency viral coat protein gpl60 (5, 6). In addition, the furin enzyme is inhibited by a1-antitrypsin genetically engineered to contain the consensus cleavage site of furin at its reactive site (7) and furin is moderately inhibited by the similarly mutated turkey ovomucoid third domain (8). However, as yet, potent naturally occurring inhibitors of the proprotein cleavage enzymes have not been identified.The neuroendocrine-specific polypeptide 7B2 was initially isolated from porcine anterior pituitary glands as a protein of ;21 kDa (9). Biosynthesis ofthe %21-kDa 7B2 protein occurs through carboxyl-terminal processing of a --27-kDa precursor protein (10,11) and only the cleaved form of 7B2 is released (10). Secretion of the 7B2 cleavage product could be regulated (10,12,13), establishing that, like PC1/PC3 and PC2, the polypeptide 7B2 is in the regulated secretory pathway. The 7B2 protein is highly conserved and widely distributed in the central nervous system and endocrine tissues (14)(15)(16) and was generated by PCR using specific primers; the 5' primer corresponded to nucleotides 107-129 with a BamHI site introduced at the 5' end and the 3' primer consisted of nucleotides 538-562 with an introduced 5' stop codon and 5' HindIII site. The recombinant 21-kDa 7B2 protein represents amino acids 1-151 of the human 7B2 protein and corresponds to the 7B2 cleavage product isolated from anterior pituitaries (9,11). The expression plasmid for the intact 27-kDa 7B2 precursor protein was constructed by replacing the =0.2-kb Kpn I-HindIII fiagment of the 21-kDa 7B2 construct by the -0.8-kb Kpn 1-HindIII fragment (encoding the carboxylterminal half of the precursor protein) of a full-length human 7B2 cDNA clone (18). Recombinant 7B2 was purified by Ni2+-NTA agarose affinity chromatography according to the instructions of the manufacturer (Qiagen, Chatsworth, CA).Preparation of PC1/PC3 and PC2 Enzymes. Active 87-kDa PC1/PC3 was purified from medium of overexpressing CHO cells as described (19). PC2 was obtained from the conditioned medium of ,BTC3 cells through immunopurification (20). One hundred milliliters of 16-h conditioned fffC3 cell culture medium (containing aprotinin at 100 jug/ml) was collected, centrifuged, and conce...
We have isolated and sequenced cDNA clones encoding the poly(A)-binding protein of Xenopus laevis oocytes. Polyclonal antiserum was raised against a fusion protein encoding 185 amino acids of the Xenopus poly(A)-binding protein. This antiserum localizes the poly(A)-binding protein to subcellular sites associated with protein synthesis; in the retina, immunoreactive protein is detected in the synthetically active inner segment of the photoreceptor but not in the transductive outer segment. Transcripts encoding the poly(A)-binding protein are present in oocytes, although no protein is detected on protein blots. In contrast, the levels of both transcripts and protein increase in development, which correlates with the observed increase in total poly(A) during Xenopus embryogenesis (N. Sagata, K. Shiokawa, and K. Yamana, Dev. Biol. 77:431-448, 1980).
The three-dimensional structure of the DNA-binding domain of the human retinoic acid receptor-beta (hRAR-beta) has been determined by nuclear magnetic resonance spectroscopy in conjunction with distance geometry, restrained molecular dynamics and iterative relaxation matrix calculations. A total of 1244 distance restraints were obtained from NOE intensities, of which 448 were intra-residue and 796 inter-residue restraints. In addition 23 chi and 30 phi dihedral angle restraints were obtained from J-coupling data. The two 'zinc-finger' regions of the 80-amino acid residue protein are followed by two alpha-helices that cross each other perpendicularly. There is a short stretch of b-sheet near the N-terminus. The alpha-helical core of the protein is well determined with a backbone root-mean-square deviation (r.m.s.d.) with respect to the average of 0.18 A and 0.37 A when the side chains of residues 31, 32, 36, 61, 62, 65 and 69 are included. The r.m.s.d. for the backbone of residues 5-80 is 0.76 A. For the first finger (residues 8-28), the r.m.s.d. of the backbone is 0.79 A. For the second finger (residues 44-62) the r.m.s.d. is 0.64 A. The overall structure is similar to that of the corresponding domain of the glucocorticoid receptor, although the C-terminal part of the protein is different. The second alpha-helix is two residues shorter and is followed by a well-defined region of extended backbone structure.
Through the use of a screening strategy designed to isolate novel cDNAs encoding proteins concerned with pituitary secretion in Xenopus laevis, we discovered clone X7365, which encodes a transmembrane protein with a signal peptide, two follistatin modules, a unique epidermal growth factor domain, and a short cytoplasmic region. RNA expression analyses indicated that the X7365 transcript is enriched in neuroendocrine tissues. lmmunohistochemical studies demonstrated that, in addition to being expressed in the optic tectum and in astrocytes in the optic nerve, the X7365 protein is concentrated in a discrete population of hypothalamic magnocellular neurons. Axons projecting to the median eminence and the neurointermediate lobe of the pituitary were also immunopositive, suggesting that X7365 functions in the regulation of magnocellular neurosecretion.
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