Proteolytic processing of presecretory proteins is required for development of biological activities in pancreatic exocrine proteins

The biological activities of pancreatic presecretory and secretory proteins synthesized in vitro were compared in studies of (a) the binding of nascent amylase to its substrate, glycogen, (b) the binding of nascent trypsinogen 1, trypsinogen 2+3, and chymotrypsinogen 1 to Sepharose-bound soybean trypsin inhibitor, and (c) the activation of nascent trypsinogen by porcine enterokinase. Nascent secretory proteins synthesized in vitro using a mRNA-dependent gel-filtered reticulocyte lysate translation system supplemented with canine pancreas rough microsomes or canine pancreas mRNA and micrococcal nuclease-treated microsomal membranes showed biological activities similar to authentic secretory proteins if oxidized glutathione was added during their synthesis. Proteins synthesized in the presence of membranes and the absence of glutathione showed significantly less biological activity due to incorrect development of conformation. Presecretory proteins synthesized in vitro with canine pancreas mRNA in the absence of microsomal membranes had little or no activity after translation in either the absence or presence of glutathione. These and previous findings (Scheele, G. A., and Jacoby, R. (1982) J. Biol. Chem. 257, 12277-12282) indicate that proteolytic removal of the NH2-terminal transport peptide is necessary to allow correct conformational development, including the formation of native disulfide bonds, which not only stabilizes the molecule but allows expression of authentic biological and probiological activity.     

Proteolytic processing of reovirus is required for adherence to intestinal M cells.

Reovirus adheres specifically to apical membranes of mouse intestinal M cells and exploits M-cell transepithelial transport activity to enter Peyer's patch mucosa, where replication occurs. Proteolytic conversion of native reovirus to intermediate subviral particles (ISVPs) occurs in the intestine, but it is not known whether conversion is essential for interaction of virus with M cells. We tested the capacity of native virions, ISVPs, and cores (that lack outer capsid proteins) to bind to intestinal epithelial cells in vivo and found that only ISVPs adhered to M cells. Thus, intraluminal conversion of native reovirus to ISVPs is a prerequisite for M-cell adherence, and outer capsid proteins unique to ISVPs (either sigma 1 or products of mu 1) mediate interaction of virus with M-cell apical membranes.     

Proteolytic processing of human zona pellucida proteins.

Formation of the egg's extracellular matrix, the zona pellucida, is critical for fertilization and development of growing embryos. Zona pellucida glycoproteins, ZP1, ZP2, and ZP3, are secreted to form an insoluble extracellular matrix surrounding mammalian eggs. All cloned mammalian zona pellucida sequences contain a furin consensus cleavage site, RX(K)/(R)R, upstream of a putative transmembrane domain, which suggests processing by an endoprotease of the furin-proprotein-convertase family. Recombinant expression of human (h) ZP1, ZP2, and ZP3 produces glycoproteins that are secreted and have migration patterns in SDS-PAGE identical to those of native human zona pellucida proteins. Because a C-terminal epitope tag that is present in the cell-associated zona proteins is, however, absent from the secreted zona proteins, secreted recombinant zona pellucida proteins lack their C-terminal regions. Three different strategies were used to explore processing events in the C-terminal region: site-directed mutagenesis of the furin cleavage site, treatment with a competitive inhibitor of all furin family members, and interference with Golgi modifications by Brefeldin A. All treatments altered the SDS-PAGE migration of recombinant hZP3, concordant with cleavage by a furin family member and Golgi glycosylation of secreted hZP3. Furthermore, cleavage of cell-associated hZP3 by exogenous furin converts the migration of cell-associated hZP3 to that of secreted hZP3. To determine whether a similar cleavage pattern exists in zona pellucida proteins that are assembled in the zona matrix, "hZP3 rescue" mouse zonae pellucidae were employed. Immunoblotting experiments revealed that hZP3, assembled and functional in the "hZP3 rescue" mouse zona pellucida, lacks the furin cleavage site, supporting the hypothesis that formation of the zona pellucida matrix involves regulated proteolysis by a member of the furin convertase family.     

Proteolytic cleavage of gram-positive beta recombinase is required for crystallization.

Beta recombinase, a DNA resolvase-invertase, catalyzes in the presence of a chromatin-associated protein such as Hbsu, DNA resolution or DNA inversion on supercoiled substrates containing two directly or inversely oriented target (six) sites. Single crystals of the beta recombinase from plasmid pSM19035 were obtained using the vapor diffusion technique with ammonium phosphate as the precipitating agent. The crystals diffracted X-rays to a maximum resolution of 2.5A. Due to proteolytic degradation during the crystallization experiment, the crystals contain only the N-terminal catalytic domain of beta recombinase corresponding to about 60% of the molecular mass of the initially assayed native protein. The proteolytic removal of the C-terminal DNA-binding domain demonstrated that protein modification can be essential to provide material suitable for X-ray analysis.     

Nucleotide sequences required for Tn3 transposition immunity.

The Tn3 transposon inserts at a reduced frequency into a plasmid already containing a copy of Tn3, a phenomenon known as transposition immunity. The cis-acting site on Tn3 responsible for immunity was mapped by deletions from each side to be within the terminal 38-base-pair sequence that is inversely repeated at the ends of Tn3. Two palindromic sequences are present in the essential part of this region. Some deletions conferred only partial immunity, and others conferred negative immunity. Multiple copies of partially immune ends conferred additional immunity. No other part of Tn3 was necessary for immunity.     

Ty3 requires yeast La homologous protein for wild-type frequencies of transposition

The Saccharomyces cerevisiae retrovirus-like element Ty3 inserts specifically into the initiation sites of genes transcribed by RNA polymerase III (pol III). A strain with a disruption of LHP1, which encodes the homologue of autoantigen La protein, was recovered in a screen for mutants defective in Ty3 transposition. Transposition into a target composed of divergent tRNA genes was decreased eightfold. In lhp1 mutants, Ty3 polyproteins were produced at wild-type levels, assembled into virus-like particles (VLPs) and processed efficiently. The amount of cDNA associated with these particles was about half the amount in a wild-type control at early times, but approached the wild-type level after 48 h of induction. Ty3 integration was examined at two genomic tRNA gene families and two plasmid-borne tRNA promoters. Integration was significantly decreased at one of the tRNA gene families, but was only slightly decreased at the second tRNA gene family. These findings suggest that Lhp1p contributes to Ty3 cDNA synthesis, but might also act at a target-specific step, such as integration.     

Ty1 proteolytic cleavage sites are required for transposition: all sites are not created equal

The retroviral protease is a key enzyme in a viral multienzyme complex that initiates an ordered sequence of events leading to virus assembly and propagation. Viral peptides are initially synthesized as polyprotein precursors; these precursors undergo a number of proteolytic cleavages executed by the protease in a specific and presumably ordered manner. To determine the role of individual protease cleavage sites in Ty1, a retrotransposon from Saccharomyces cerevisiae, the cleavage sites were systematically mutagenized. Altering the cleavage sites of the yeast Ty1 retrotransposon produces mutants with distinct retrotransposition phenotypes. Blocking the Gag/PR site also blocks cleavage at the other two cleavage sites, PR/IN and IN/RT. In contrast, mutational block of the PR/IN or IN/RT sites does not prevent cleavage at the other two sites. Retrotransposons with mutations in each of these sites have transposition defects. Mutations in the PR/IN and IN/RT sites, but not in the Gag/PR site, can be complemented in trans by endogenous Ty1 copies. Hence, the digestion of the Gag/PR site and release of the protease N terminus is a prerequisite for processing at the remaining sites; cleavage of PR/IN is not required for the cleavage of IN/RT, and vice versa. Of the three cleavage sites in the Gag-Pol precursor, the Gag/PR site is processed first. Thus, Ty1 Gag-Pol processing proceeds by an ordered pathway.     

Proteolytic events in the processing of secreted proteins in fungi.

Secreted heterologous proteins have been found to be produced much less efficiently by fungi than secreted homologous ones. This could be due, at least in part, to proteolytic cleavage by site-specific endoproteases of the secretory pathway, similar to the yeast KEX2 protease and the mammalian dibasic endoproteinases found in secretory pathways. Mature secreted fungal proteins may be protected from such cleavage due to the absence of cleavable sites in exposed regions. A comparison of the dipeptide distributions of 33 secreted and 34 cytoplasmic proteins from fungal producers of extracellular enzymes indicated a significant bias for some doublets, including the basic dipeptides Lys-Arg, Arg-Arg and Arg-Lys which have also been demonstrated to be KEX2 substrates. Other combinations were also found to be rare in secreted proteins, which could indicate either a broader specificity of the considered endopeptidase, or the presence either in the secretory organelles or among the secreted proteins of additional proteases with different specificities. Experimental evidence that the Lys-Arg site is processed in Tolypocladium geodes was provided by cloning a synthetic prosequence upstream of a phleomycin resistance (Sh ble) gene and analyzing the N-terminus of the corresponding protein purified from the culture supernatant. This system also provides a tool for further studies of specific proteases of fungi.     

NOP3 is an essential yeast protein which is required for pre-rRNA processing

The four nucleolar proteins NOP1, SSB1, GAR1, and NSR1 of Saccharomyces cerevisiae share a repetitive domain composed of repeat units rich in glycine and arginine (GAR domain). We have cloned and sequenced a fifth member of this family, NOP3, and shown it to be essential for cell viability. The NOP3 open reading frame encodes a 415 amino acid protein with a predicted molecular mass of 45 kD, containing a GAR domain and an RNA recognition motif. NOP3-specific antibodies recognize a 60-kD protein by SDS-PAGE and decorate the nucleolus and the surrounding nucleoplasm. A conditional lethal mutation, GAL::nop3, was constructed; growth of the mutant strain in glucose medium represses NOP3 expression. In cells depleted of NOP3, production of cytoplasmic ribosomes is impaired. Northern analysis and pulse-chase labeling indicate that pre-rRNA processing is inhibited at the late steps, in which 27SB pre-rRNA is cleaved to 25S rRNA and 20S pre-rRNA to 18S rRNA.     

Proper processing of avian sarcoma/leukosis virus capsid proteins is required for infectivity

The formation of the mature carboxyl terminus of CA in avian sarcoma/leukemia virus is the result of a sequence of cleavage events at three PR sites that lie between CA and NC in the Gag polyprotein. The initial cleavage forms the amino terminus of the NC protein and releases an immature CA, named CA1, with a spacer peptide at its carboxyl terminus. Cleavage of either 9 or 12 amino acids from the carboxyl terminus creates two mature CA species, named CA2 and CA3, that can be detected in avian sarcoma/leukemia virus (R. B. Pepinsky, I. A. Papayannopoulos, E. P. Chow, N. K. Krishna, R. C. Craven, and V. M. Vogt, J. Virol. 69:6430-6438, 1995). To study the importance of each of the three CA proteins, we introduced amino acid substitutions into each CA cleavage junction and studied their effects on CA processing as well as virus assembly and infectivity. Preventing cleavage at any of the three sites produced noninfectious virus. In contrast, a mutant in which cleavage at site 1 was enhanced so that particles contained CA2 and CA3 but little detectable CA1 was infectious. These results support the idea that infectivity of the virus is closely linked to proper processing of the carboxyl terminus to form two mature CA proteins.