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Located within the perisinusoidal space and surrounded by extracellular matrix, hepatic stellate cells (HSC) undergo phenotypic trans-differentiation called "myofibroblastic activation" in liver fibrogenesis. This study investigated the regulation of interleukin-1 (IL-1alpha) on expression of matrix metalloproteinases (MMPs) by HSC grown in three-dimensional extracellular matrix and the role of MMPs in HSC activation. To recapitulate the in vivo "quiescent" state of HSC, the isolated rat HSC were grown in three-dimensional Matrigel or type I collagen. Stimulation with IL-1alpha caused robust induction of pro-MMP-9 (the precursor of matrix metalloproteinase-9) when HSC were cultured in these matrices. IL-1alpha induced a conversion of the pro-MMP-9 to the active form only when the cells were in type I collagen. In collagen lattices, IL-1alpha provoked activation of HSC with induction of MMP-13, MMP-3, and breakdown of the matrix. The HSC activation was completely prevented by a treatment of the cells with tissue inhibitor of metalloproteinase-1 or deprivation of MMP-9. Once fully activated, HSC failed to express MMP-9 and showed attenuated induction of MMP-13 and MMP-3. Further, we demonstrated colocalization of alpha-smooth muscle actin and MMP-9 in a subpopulation of HSC in human fibrotic liver tissues. Thus, this study provides a novel model to enlighten the role of MMPs, particularly that of MMP-9, in HSC activation regulated by a specific cytokine in liver fibrogenesis.

Proteolytic processing of amyloid precursor protein generates beta-amyloid (Abeta) peptides that are deposited in senile plaques in brains of aged individuals and patients with Alzheimer's disease. Presenilins (PS1 and PS2) facilitate the final step in Abeta production, the intramembranous gamma-secretase cleavage of amyloid precursor protein. Biochemical and pharmacological evidence support a catalytic or accessory role for PS1 in gamma-secretase cleavage, as well as a regulatory role in select membrane protein trafficking. In this report, we demonstrate that PS1 is required for maturation and cell surface accumulation of nicastrin, an integral component of the multimeric gamma-secretase complex. Using kinetic labeling studies we show that in PS1(-/-)/PS2(-/-) cells nicastrin fails to reach the medial Golgi compartment, and as a consequence, is incompletely glycosylated. Stable expression of human PS1 restores these deficiencies in PS1(-/-) fibroblasts. Moreover, membrane fractionation studies show co-localization of PS1 fragments with mature nicastrin. These results indicate a novel chaperone-type role for PS1 and PS2 in facilitating nicastrin maturation and transport in the early biosynthetic compartments. Our findings are consistent with PS1 influencing gamma-secretase processing at multiple steps, including maturation and intracellular trafficking of substrates and component(s) of the gamma-secretase complex.

To further our understanding of the structure and function of phosphodiesterases of the newly identified family of high affinity cAMP-specific phosphodiesterases (PDE7), we identified and characterized the isozyme expressed in human skeletal muscle and the protein product of the previously isolated isozyme HCP1 (designated HSPDE7A1). We report the isolation of a cDNA encoding the full-length skeletal muscle isoform of human PDE7A (HSPDE7A2). The DNA sequence of this skeletal muscle cDNA indicates thatPDE7A2 is a novel 5′ splice variant of PDE7Aencoding an isoform with a novel, hydrophobic N terminus. The 456-amino acid PDE7A2 protein is detected on Western blots as a band with an apparent mobility of 50 kDa. PDE7A2 is a high affinity cAMP-specific PDE (K m = 0.1 μm), which is localized to particulate cellular fractions. The PDE7A1 (HCP1) isozyme is detected on Western blots as a band with an apparent mobility of 57 kDa, demonstrating that the previously isolated HCP1cDNA encodes the full-length PDE7A1 protein. The even distribution of PDE7A mRNA among fetal tissues and the relative abundance of its two mRNAs strongly suggest that the expression ofPDE7A is regulated throughout development.

Human immunodeficiency virus type 1 (HIV-1) protease optimally catalyzes in the pH range of 4-6 in contrast to nearly all of the other eukaryotic aspartic proteases, which catalyze best in the pH range of 2-4. A possible structural reason for the higher optimal pH of HIV-1 protease is the absence of a hydrogen bond to the carboxyl group of active-site Asp25, which is nearly universally present in others. To investigate this hypothesis, we have mutated residue 28 in HIV-1 protease from alanine to serine. Both the wild-type and the mutant A28S enzymes have been overexpressed in Escherichia coli using a chemically synthesized gene and purified for a comparative study in enzyme kinetics. The kcat and Km values were determined by a radiometric assay for the wild-type enzyme from pH 3.2 to 7.0, and for the mutant enzyme from pH 3.2 to 6.0. The low pK values of the active site of the free enzyme, pKe1, are 3.3 and 3.4 for the wild-type and mutant enzymes, respectively. The low pK values of the active site of the enzyme bound to substrate, pKes1, are 5.1 and 4.3 for the wild-type and mutant enzymes, respectively. The high pK values of the free enzyme, pKe2, are 6.8 and 5.6, and the corresponding ones for the substrate-bound enzyme, pKes2, are 6.9 and 6.0 for the wild-type and mutant enzymes, respectively. The lowering of pK values in mutant HIV-1 protease indicates that the hydroxyl group of Ser28 forms a new hydrogen bond to active-site Asp25 to increase its acidity.

A cDNA clone, which contained the complete rhizopuspepsin structure and the putative proregion, was placed in three different Escherichia coli expression vectors for the synthesis of rhizopuspepsinogen (Rpg). Recombinant Rpgs which were expressed in the cytosol of E. coli as inclusion bodies (cRpg and tRpg) were not active. After solubilization in 6 M urea and refolding by rapid dilution, both of these Rpgs were purified to homogeneity. The third zymogen, pRpg, which was secreted to the periplasmic space of E. coli with an omp leader, was fully active and also was purified. The expression level of pRpg was higher (over 40 mg/liter culture) than that of cRpg (about 1.5 mg/liter culture). Amino-terminal sequence analysis of the zymogens revealed that cRpg and pRpg contain 40 and 51 residues of prosequence, respectively. tRpg, which was expressed under the control of T7 promoter, was synthesized at 500 mg/liter culture and was purified at 50 mg/liter culture. This zymogen contained, in addition to 51 residues of proregion, 16 residues inherited from the expression vector construction. All of these Rpgs spontaneously converted to rhizopuspepsin in solutions of pH less than 5. Each of the conversions was associated with a change of molecular weight as monitored in sodium dodecyl sulfate-polyacrylamide electrophoresis. At least one intermediate of conversion was observed in the pH range of 2 to 3 for both the cRpg and pRpg zymogens. For pRpg and tRpg, kinetic data demonstrated that the Rpg to rhizopuspepsin conversion was accomplished by a first order, unimolecular reaction at pH 2. The first order kinetic constants in this pH at 15 degrees C were 1.1 and 2.4 min-1 for pRpg and tRpg, respectively. The activation rate decreased as pH was raised above pH 2. At pH greater than 3.0, rhizopuspepsin-catalyzed, second-order activation also takes place. Consequently, the recombinant Rpgs are activated by either of two cleavage mechanisms as is the case for pepsinogen. These results also support the hypothesis that Rpg is synthesized in Rhizopus chinensis as a zymogen. Rpg in the host fungus is probably activated by an acid environment of pH less than 5 in the secretory granules to become rhizopuspepsin before secretion.