We describe a novel 30-kDa secretory protein, Acrp30 (adipocyte complement-related protein of 30 kDa), that is made exclusively in adipocytes and whose mRNA is induced over 100-fold during adipocyte differentiation. Acrp30 is structurally similar to complement factor C1q and to a hibernation-specific protein isolated from the plasma of Siberian chipmunks; it forms large homo-oligomers that undergo a series of post-translational modifications. Like adipsin, secretion of Acrp30 is enhanced by insulin, and Acrp30 is an abundant serum protein. Acrp30 may be a factor that participates in the delicately balanced system of energy homeostasis involving food intake and carbohydrate and lipid catabolism. Our experiments also further corroborate the existence of an insulin-regulated secretory pathway in adipocytes.
Abstract. We have identified and characterized Cab45, a novel 45-kD protein from mouse 3T3-L1 adipocytes. Cab45 is ubiquitously expressed, contains an NH2-terminal signal sequence but no membrane-anchor sequences, and binds Ca 2÷ due to the presence of six EFhand motifs. Within the superfamily of calcium-binding proteins, it belongs to a recently identified group of proteins consisting of Reticulocalbin (Ozawa, M., and T. Muramatsu. 1993. J. Biol. Chem. 268:699-705) and ERC 55 (Weis, K., G. Griffiths, and A.I. Lamond. 1994. J. Biol. Chem. 269:19142-19150), both of which share significant sequence homology with Cab45 outside the EF-hand motifs. In contrast to reticulocalbin and ERC-55 which are soluble components of the endoplasmic reticulum, Cab45 is a soluble protein localized to the Golgi.Cab45 is the first calcium-binding protein localized to the lumenal portion of a post-ER compartment; Cab45 is also the first known soluble protein resident in the Golgi lumen. Cab45 can serve as a model protein to determine the mechanism of retention of soluble proteins in the Golgi compartment.
The complete protein precursor of human kidney renin has been determined from the sequence of cloned genomic DNA. The gene spans 12 kilobases of DNA and is interrupted by eight intervening sequences. The nine regions (exons) encoding the protein were mapped with a mouse renin cDNA probe, synthetic oligonucleotide probes, and by hybridization of genomic restriction fragments to a 1600-nucleotide human kidney mRNA. The predicted 403-amino acid preprorenin consists of mature renin and a 66-residue amino-terminal prepropeptide. The DNA sequence 5' to the first exon indicates the location of a transcriptional promoter (T-A-T-A-A-A) for a mRNA encoding preprorenin. An additional transcriptional promoter site is located within the first intron, which, if used, would express a shortened nonsecreted prorenin. The structure of the human renin gene is similar to that of human pepsinogen, a closely related aspartyl protease enzyme. This observation suggests that renin and pepsinogen have a common evolutionary origin.Renin is an endocrine hormone catalyzing the first step in a cascade of factors that modulate arteriole blood pressure. It hydrolyzes a single peptide bond in the circulating globulin, angiotensinogen, releasing the amino-terminal decapeptide angiotensin I. The absolute specificity of renin is in contrast to other aspartyl proteases, which act on a broad range of substrates (1). The characterization of this specificity is of considerable pharmaceutical and medical interest. However, detailed biochemical analysis of the protein has been limited because the hormone is produced in such small amounts by its known physiological source, the juxtaglomerular cells of the kidney cortex. Renin purified from human tissue has shown variations in molecular weight and amino acid composition (2-4).We report here the isolation and sequence analysis of the human renin gene.EXPERIMENTAL PROCEDURES Plaque Screening. A library of bacteriophage Charon 4A containing human fetal DNA (5) was grown in Escherichia coli strain LE392 (6). Nitrocellulose filter (Schleicher & Schuell) replicas of -300,000 plaques were incubated in 2x NaCl/Cit (lx NaCl/Cit is 0.15 M NaCl/0.015 M Na citrate)/0.1% NaDodSO4/5x Denhardt's solution (lx Denhardt's solution is 0.02% polyvinylpyrrolidone/Ficoll (Pharmacia)/bovine serum albumin) at 55°C overnight and then hybridized (106 cpm per filter) to a mouse renin probe [labeled to greater than 108 cpm/,g of DNA by nick-translation (7) in the presence of [a-32P]dATP and dCTP (New England Nuclear)] at 55°C for 24 hr in 2x NaCl/Cit/0.1% NaDodSO4/1x Denhardt's solution/10% dextran sulfate (Pharmacia). Filters were washed at 50°C in 0.1x NaCl/Cit/0.1% NaDodSO4 prior to autoradiography. Positive plaques were purified by standard methods (8). Cloning procedures were according to National Institutes of Health guidelines.Hybridization to Filter-Bound mRNA. RNA was purified (9) from human kidneys (obtained immediately postmortem or from surgical nephrectomy) and the poly(A)+ mRNA was fractionated using poly(U)-Se...
A model has been constructed using computer graphics for human renin based on the sequence derived from that of the gene and the 3-dimensional structure defined at high resolution for other homologous aspartic proteinases. Human renin can adopt a 3-dimensional structure close to that of other aspartic proteinases, in which amino acids corresponding to intron-exon junctions in the gene are at surface regions in the 3-dimensional structure. As expected, the essential catalytic residues are retained and the nearby residue 304 is alanine as in the mouse sequence, supporting the idea that Asp 304 of other aspartic proteinases may contribute to the low pH of their optimal activity. There are interesting differences at subsite S; which may contribute to the specificity of human renin. Certain residues at the surface of the enzyme adjacent to the active site cleft are unique to renins and may play a role in recognition and binding of angiotensinogen.
The UV light inducibility of the uvrB operon of Escherichia coli K-12 was previously demonstrated by exploiting a strain in which the gene for the enzyme beta-galactosidase was inserted into the uvrB operon. This insert is now shown to be located within the structural gene for the uvrB enzyme, leaving the regulatory sequences of the operon intact. Analyses to quantitate the induction of this system show that derepression of the operon is first detectable 5 min after UV exposure, with the rate of synthesis increasing to four to six times the uninduced rate during the subsequent 30 min. Induction is unaffected by mutations in other components of nucleotide excision repair. The control of uvrB was found to result from direct repression by the lexA gene product, with the recA gene product playing an indirect role. Nucleotide excision repair thus seems to be part of the SOS response.
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