Proteolytic precursor processing in the biosynthesis of mitochondria

Essentially all polypeptides synthesized in the cytoplasm and imported into either the matrix or into the inner or outer membrane of mitochondria are made as larger molecular weight precursors. All known examples of in vivo or in vitro synthesized precursors are summarized. Little information on the nature of the proteolytic enzymes involved in the processing of the larger precursor polypeptides exists. The biosynthesis of rat liver cytochrome c oxidase is discussed in detail. In contrast to reported data, the cytoplasmic subunits of rat liver cytochrome c oxidase are synthesized as larger molecular weight precursors and not as a polyprotein. Precursors to subunits IV and V show an extra-peptide sequence of about 3000 daltons. Evidence against the existence of a polyprotein precursor was also obtained, when messenger RNAs for the individual subunits IV and V were isolated and analyzed in respect to their size. A length of 990 +/- 80 and 830 +/- 70 nucleotides was estimated for the poly(A)+-RNA of cytochrome c oxidase subunits IV and V, respectively. In experiments on the site of synthesis, it was found that cytochrome c oxidase subunits IV and V are made on free, loosely and tightly membrane-bound polyribosomes.     

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.     

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 activity in the insulin receptor

When a highly purified preparation of rat liver insulin receptor is incubated with trypsin, the receptor develops hydrolytic activity towards N alpha-benzoyl-L-arginine ethyl ester, N alpha-p-tosyl-L-arginine methyl ester, and N alpha-benzoyl-DL-arginine-p-nitroanilide, (compounds which are synthetic substrates of trypsin). The activity toward N alpha-benzoyl-DL-arginine-p-nitroanilide is inhibited by soybean trypsin inhibitor but not by N alpha-p-tosyl-L-lysil chloromethyl ketone. These data are consistent with the presence of proteolytic activity in the insulin receptor specific for the bonds whose carbonyl functions are provided by arginine but not by lysine. Furthermore we found that the esterase activity toward N alpha-benzoyl-L-arginine ethyl ester in the presence of trypsin is enhanced by insulin, and that the concentration of insulin that produced the half maximum stimulation is of the same magnitude as the dissociation constant for the soluble receptor. These data suggest that the insulin receptor is a zymogen, activated by trypsin, and based on its specific activity, may be the protease which releases a peptide chemical mediator of intracellular action of insulin.     

Proteolytic processing of Ty3 proteins is required for transposition.

Ty3 is a retroviruslike element found in Saccharomyces cerevisiae. It encodes GAG3 and GAG3-POL3 polyproteins which are processed into mature proteins found in the Ty3 viruslike particle. In this study, the region encoding a protease that is homologous to retroviral aspartyl proteases was identified and shown to be required for production of mature Ty3 proteins and transposition. The Ty3 protease has the Asp-Ser-Gly consensus sequence found in copia, Ty1, and Rous sarcoma virus proteases, rather than the Asp-Thr-Gly found in most retroviral proteases. The Asp-Ser-Gly consensus is flanked by residues similar to those which flank the active sites of cellular aspartyl proteases. Mutations were made in the Ty3 active-site sequence to examine the role of the protease in Ty3 particle maturation and to test the functional significance of the Ser active-site variant in the consensus sequence. Mutation of the active-site Asp blocked processing of Gag3 and Gag3-Pol3 and allowed identification of a GAG3-POL3 polyprotein. This protein was turned over rapidly in cells expressing the mutant Ty3. Changing the active-site Ser to Thr caused only a modest reduction in the levels of certain Ty3 proteins. Five putative cleavage sites of this protease in Ty3 GAG3 and GAG3-POL3 polyproteins were defined by amino-terminal sequence analysis. The existence of an additional protein(s) of unknown function, encoded downstream of the protease-coding region, was deduced from the positions of these amino termini and the sizes of known Ty3 proteins. Although Ty3 protease cleavage sites do not correspond exactly to known retroviral protease cleavage sites, there are similarities. Residues P3 through P2' in the regions encompassing each of the five sites are uncharged, and no P1 position is occupied by an amino acid with a branched beta carbon.     

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 the insulin receptor beta subunit is associated with insulin-induced receptor down-regulation

This report describes the use of an antibody directed against the carboxyl terminus of the insulin receptor beta subunit to assess the fate of the insulin receptor protein over the time course of insulin-induced receptor down-regulation. The insulin receptor beta subunit is lost from the cellular membranes of insulin-treated 3T3-C2 fibroblasts with a time course superimposable with the insulin-induced loss of cellular insulin binding activity. Concomitant with the time-dependent loss of the intact beta subunit from the membranes, a 61,000-Da fragment of the insulin receptor beta subunit accumulates in the cytosol of the cells in a time-dependent manner. The insulin-induced loss of the intact beta subunit from the cellular membranes is inhibited by cycloheximide. Chloroquine and the thiol protease inhibitors leupeptin and E-64 inhibit the insulin-induced loss of the intact beta subunit from the membranes and induce an accumulation of the intact subunit in the membranes. However, in the presence of leupeptin, E-64, or chloroquine, the insulin-induced loss of insulin binding activity occurs normally. These data indicate that down-regulation results in the loss of the intact beta subunit from the cellular membranes with the production of a fragment of the beta subunit in the cytosol. The protease responsible for the generation of the fragment is a thiol protease which requires acidic conditions. Since the insulin-induced proteolysis of the beta subunit can be totally inhibited under conditions where the insulin-induced loss of insulin binding activity proceeds normally, the proteolysis of the beta subunit is a process which is separate and distinguishable from the insulin-induced loss of insulin binding activity.     

Proteolytic processing in development and leukemogenesis

There are now numerous examples in the hematopoietic system of genes that are critical for normal hematopoietic development, but when mutated, rearranged, or overexpressed, contribute to leukemogenesis. Two papers in this issue of Cell provide a fascinating twist on this paradigm, and suggest that proteolytic processing of certain of these genes plays an important role both in development and in leukemogenesis. These findings also suggest the possibility that proteases may be therapeutic targets in leukemia.     

Proteolytic processing of the astrovirus capsid

To further characterize the nature of proteolytic processing of the astrovirus capsid, we infected Caco-2 cells with a high multiplicity of astrovirus without trypsin in the presence of 5 to 10% fetal calf serum. These infections were characterized by pulse-chase labeling with [35S]Smethionine, electron microscopy, gel electrophoresis of purified viral particles, and analysis of infectivity of such particles with and without added trypsin. Pulse-chase experiments showed that the astrovirus capsid protein was initially translated as an approximately 87-kDa protein. The 87-kDa capsid protein was rapidly converted intracellularly to a 79-kDa form which was found in smaller amounts in the cell supernatant. Purification by differential centrifugation yielded particles that appeared quite similar to trypsin-grown astrovirus particles by negatively stained electron microscopy. These particles were antigenically distinct from trypsin-treated virions as demonstrated by their various reactions with monoclonal antibodies in a solid-phase immunoassay. The purified trypsin-free particles were mainly composed of the 79-kDa capsid protein which was found to have an amino terminus at residue 71 of the entire open reading frame 2 (ORF2) product. The cleavage site was identified in a highly conserved region of the astrovirus ORF2 product. These trypsin-free particles were minimally infectious in cultured Caco-2 cells but became highly infectious (10(5)-fold increase) after trypsin but not chymotrypsin treatment. This trypsin-enhanced infectivity correlated with conversion of the 79-kDa capsid protein to three smaller peptides of approximately 34, 29, and 26 kDa.     

Proteolytic processing and physiological regulation

Proteolytic processing is a common and effective mechanism of physiological regulation. The basic principle is a conformational change induced in the protein precursor by the post-translational proteolytic cleavage of a specific peptide bond. The extension of earlier studies of model zymogens to more complex systems of physiological regulation, using methods of both protein chemistry and molecular biology, has enormously extended knowledge of the repertoire of proteolytic processing reactions and has contributed significantly to current studies of the structure, domain organization and evolution of proteins.     

Proteolytic processing and regulation.

Many proteins, particularly proteolytic enzymes, protein hormones and neuropeptides are synthesized as inactive precursors that undergo posttranslational processing by proteolytic enzymes. The roots of current knowledge go back to the early observations of the activation of zymogens. A major advance followed the discovery of the polypreprotein, pre-pro-opiomelanolcortin and of proinsulin, and the characterization of the mammalian processing prohormone enzyme as members of the multidomain yeast kexin family. More recent applications of methods of molecular biology have greatly advanced our understanding of the nature and mode of action of these proteases.     

Proteolytic processing of turnip yellow mosaic virus replication proteins and functional impact on infectivity

Turnip yellow mosaic virus (TYMV), a positive-strand RNA virus belonging to the alphavirus-like supergroup, encodes its nonstructural replication proteins as a 206K precursor with domains indicative of methyltransferase (MT), proteinase (PRO), NTPase/helicase (HEL), and polymerase (POL) activities. Subsequent processing of 206K generates a 66K protein encompassing the POL domain and uncharacterized 115K and 85K proteins. Here, we demonstrate that TYMV proteinase mediates an additional cleavage between the PRO and HEL domains of the polyprotein, generating the 115K protein and a 42K protein encompassing the HEL domain that can be detected in plant cells using a specific antiserum. Deletion and substitution mutagenesis experiments and sequence comparisons indicate that the scissile bond is located between residues Ser879 and Gln880. The 85K protein is generated by a host proteinase and is likely to result from nonspecific proteolytic degradation occurring during protein sample extraction or analysis. We also report that TYMV proteinase has the ability to process substrates in trans in vivo. Finally, we examined the processing of the 206K protein containing native, mutated, or shuffled cleavage sites and analyzed the effects of cleavage mutations on viral infectivity and RNA synthesis by performing reverse-genetics experiments. We present evidence that PRO/HEL cleavage is critical for productive virus infection and that the impaired infectivity of PRO/HEL cleavage mutants is due mainly to defective synthesis of positive-strand RNA.     

Proteolytic degradation of insulin and glucagon.

The degradation of insulin and glucagon by a highly purified enzyme isolated from rat skeletal muscle was investigated. A sensitive assay for proteolytic degradation of insulin and glucagon using fluorescamine to detect an increase in primary amine groups was established. As measured by an increase in fluorescamine reactive materials, insulin was rapidly degraded by this highly purified enzyme without requiring initial disulfide cleavage. Associated with the increase in fluorescamine reactive materials was a decrease in immunoassayable insulinmglucagon wal also proteolytically degraded by this enzyme but a number of other peptides and proteins including proinsulin, and A and B chains of insulin were not degraded. Thus, we have demonstrated that insulin (and glucagon) can be proteolytically degraded by an enzyme isolated from an insulin sensitive tissue, skeletal muscle. Proteolytic degradation by this enzyme requires the intact insulin molecule rather than separate A and B chains.     

Proteolytic processing and translation initiation: two independent mechanisms for the expression of the Sendai virus Y proteins

The four Sendai virus C-proteins (C', C, Y1, and Y2) represent an N-terminal nested set of non-structural proteins whose expression modulates both the readout of the viral genome and the host cell response. In particular, they modulate the innate immune response by perturbing the signaling of type 1 interferons. The initiation codons for the four C-proteins have been mapped in vitro, and it has been proposed that the Y proteins are initiated by ribosomal shunting. A number of mutations were reported that significantly enhanced Y expression, and this was attributed to increased shunt-mediated initiation. However, we demonstrate that this arises due to enhanced proteolytic processing of C', an event that requires its very N terminus. Curiously, although Y expression in vitro is mediated almost exclusively by initiation, Y proteins in vivo can arise both by translation initiation and processing of the C' protein. To our knowledge this is the first example of two apparently independent pathways leading to the expression of the same polypeptide chain. This dual pathway explains several features of Y expression.     

Proteolytic processing and oligomerization of bacteriophage-derived endosialidases

Bacteriophages infecting the neuroinvasive pathogen Escherichia coli K1 require an endosialidase to penetrate the polysialic acid capsule of the host. Sequence information is available for the endosialidases endoNE, endoNF, and endoN63D of the K1-specific phages phi K1E, phi K1F, and 63D, respectively. The cloned sequences share a highly conserved catalytic domain but differ in the length of the N- and C-terminal parts. Although the expression of active recombinant enzyme succeeded in the case of endoNE, it failed for endoNF. Protein alignments of all three endosialidase sequences gave rise to the assumption that inactivity of the cloned endoNF is caused by a C-terminal truncation. By reinvestigation of the respective gene locus in the phi K1F genome, we identified an extended open reading frame of 3195 bp, encoding a 119-kDa protein. Full-length endoNF contains the C-terminal domain conserved in all endosialidases, which may act as an intramolecular chaperone. Comparative studies carried out with endoNE and endoNF demonstrate that endosialidases are proteolytically processed, releasing the C-terminal domain. Using a mutational approach in combination with protein analytical techniques we demonstrate that (i) the C-terminal domain is a common feature of endosialidases and other tail fiber proteins; (ii) the integrity of the C-terminal domain and its presence in the nascent protein are crucial for the formation of active enzymes; (iii) proteolytic processing is not essential for enzymatic activity; and (iv) functional folding is a prerequisite for trimerization of endoNF.