Cleavage of a viral polyprotein by a cellular proteolytic activity.

The 200,000-dalton polyprotein encoded by the bottom component RNA of cowpea mosaic virus was synthesized in rabbit reticulocyte lysates, and this in vitro-synthesized protein was isolated from the lysate reaction mixture by sucrose density gradient centrifugation. Incubation of the isolated polyprotein with buffer caused no change in the protein, but incubation with reticulocyte lysates or with fractionated lysate proteins resulted in cleavage of the protein into the expected cleavage products (32,000- and 170,000-dalton proteins). This finding indicated that reticulocytes contain a proteolytic activity that is needed for the primary cleavage reaction. A cleavage assay in which we used partially purified preparations showed that cleavage was an ATP-dependent reaction.     

Identification of the primary translation product of atrial natriuretic peptide receptor mRNA in a cell-free system using anti-receptor antiserum

The primary translation product of mRNA encoding atrial natriuretic peptide (ANP) receptor has been shown to have an Mr of 58,000. Poly(A)+ RNA was isolated from the bovine kidney and lung and translated in a rabbit reticulocyte lysate system containing [35S]methionine. Immunoprecipitation of the labeled translation products, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography, identified a 58-kDa protein as the primary translation product which is the unglycosylated precursor to be processed to the glycosylated mature 70-kDa form found in the plasma membranes. The result lends strong support to our previous proposal that mature ANP receptor is composed of two disulfide-linked 70-kDa subunits, eliminating the possibility that the two 70-kDa subunits arise from a larger 140-kDa precursor by proteolytic cleavage.     

Hu proteins regulate polyadenylation by blocking sites containing U-rich sequences

A recent genome-wide bioinformatic analysis indicated that 54% of human genes undergo alternative polyadenylation. Although it is clear that differential selection of poly(A) sites can alter gene expression, resulting in significant biological consequences, the mechanisms that regulate polyadenylation are poorly understood. Here we report that the neuron-specific members of a family of RNA-binding proteins, Hu proteins, known to regulate mRNA stability and translation in the cytoplasm, play an important role in polyadenylation regulation. Hu proteins are homologs of the Drosophila embryonic lethal abnormal visual protein and contain three RNA recognition motifs. Using an in vitro polyadenylation assay with HeLa cell nuclear extract and recombinant Hu proteins, we have shown that Hu proteins selectively block both cleavage and poly(A) addition at sites containing U-rich sequences. Hu proteins have no effect on poly(A) sites that do not contain U-rich sequences or sites in which the U-rich sequences are mutated. All three RNA recognition motifs of Hu proteins are required for this activity. Overexpression of HuR in HeLa cells also blocks polyadenylation at a poly(A) signal that contains U-rich sequences. Hu proteins block the interaction between the polyadenylation cleavage stimulation factor 64-kDa subunit and RNA most likely through direct interaction with poly(A) cleavage stimulation factor 64-kDa subunit and cleavage and polyadenylation specificity factor 160-kDa subunit. These studies identify a novel group of mammalian polyadenylation regulators. Furthermore, they define a previously unknown nuclear function of Hu proteins.     

A multicomponent complex is required for the AAUAAA-dependent cross-linking of a 64-kilodalton protein to polyadenylation substrates.

A 64-kilodalton (kDa) polypeptide that is cross-linked by UV light specifically to polyadenylation substrate RNAs containing a functional AAUAAA element has been identified previously. Fractionated HeLa nuclear components that can be combined to regenerate efficient and accurate polyadenylation in vitro have now been screened for the presence of the 64-kDa protein. None of the individual components contained an activity which could generate the 64-kDa species upon UV cross-linking in the presence of substrate RNA. It was necessary to mix two components, cleavage stimulation factor and specificity factor, to reconstitute 64-kDa protein-RNA cross-linking. The addition of cleavage factors to this mixture very efficiently reconstituted the AAUAAA-specific 64-kDa protein-RNA interaction. The 64-kDa protein, therefore, is present in highly purified, reconstituted polyadenylation reactions. However, it is necessary to form a multicomponent complex to efficiently cross-link the protein to a substrate RNA.     

Isolation of the messenger RNA for 8S DNA ligase in early developing axolotl egg and its cell free translation

A new DNA ligase activity is expressed when the Axolotl eggs enter cleavage. The messenger RNA can be labelled by [3H] uridine thereby indicating its de novo synthesis. This new genetic expression is occurring just before cleavage and is the earliest found during Amphibian development. The newly synthesized [3H] mRNA can be translated in vitro in the rabbit reticulocyte lysate system. The resulting product is a 160 K protein specifically immunoprecipitated with the antiserum directed against 8S DNA ligase. This in vitro translated polypeptide exhibits 8S DNA ligase activity specific of activated or fertilized eggs but does not display 6S DNA ligase activity of non activated eggs.     

Inhibition of cleavage of a plant viral polyprotein by an inhibitor activity present in wheat germ and cowpea embryos

In rabbit reticulocyte lysate, the bottom component RNA of cowpea mosaic virus directs the synthesis of a 200,000-molecular-weight precursor protein (200K protein) that is cleaved during synthesis by a reticulocyte enzyme to form a 32K protein and a 170K protein. Cleavage of the 200K protein was found to be effectively inhibited by inhibitor activity in wheat germ and cowpea embryo extracts. The inhibitor was nondialyzable, precipitatable by ammonium sulfate, and partially stable at high temperatures. The activity appeared to be specific in that it caused no inhibition of the secondary cleavage reactions (cleavage of the 170K protein) at concentrations that were sufficient to cause complete inhibition of the primary cleavage reaction (cleavage of the 200K protein).     

Characterization of structural domains of the human epidermal growth factor receptor obtained by partial proteolysis

Partial cleavage with trypsin has been used to study the structure of the epidermal growth factor (EGF) receptor purified from human carcinoma cells. Following affinity labeling of the receptor with 125I-EGF or the ATP analogue 5'-p-fluorosulfonyl benzoyl[14C]adenosine, metabolic labeling with [35S]methionine, [3H]glucosamine, or [32P]orthophosphate, or in vitro autophosphorylation with [gamma-32P]ATP, tryptic cleavage defines the following three regions of the 180-kDa receptor protein: 1) a 125-kDa trypsin-resistant domain which contains sites of glycosylation, EGF binding, and an EGF-specific threonine phosphorylation site; 2) an adjacent 40-kDa fragment which contains serine and threonine phosphorylation sites and is further cleaved to a 30-kDa trypsin-resistant domain; and 3) a terminal 15-kDa portion of the receptor that contains the sites of tyrosine phosphorylation and is degraded to small fragments in the presence of trypsin. Both the 125- and 40-kDa regions of the EGF receptor appear to be required for receptor-associated protein kinase activity since separation of these regions by tryptic cleavage abolishes this activity, and both regions are specifically labeled with an ATP affinity analogue, suggesting that both are involved in ATP binding. Additional 63- and 48-kDa phosphorylated fragments are generated upon trypsin treatment of EGF receptor from EGF-treated cells. The potential usefulness of partial tryptic cleavage in studying the EGF receptor and the possible biological function of the 30-kDa trypsin-resistant fragment of the receptor are discussed.     

The origin of multiple polypeptides of molecular weight below 110 000 encoded by tobacco mosaic virus RNA in the messenger-dependent rabbit reticulocyte lysate

Multiple polypeptides encoded by tobacco mosaic virus (TMV) RNA in the messenger-dependent rabbit reticulocyte lysate are not attributable to contaminating 3′-coterminal RNA fragments, multiple leaky termination codons or endonuclease activity opening-up legitimate or spurious internal initiation sites. Quantitative analysis of polypeptides encoded over a range of added RNA concentrations from 0.09 μg·ml^?1 to 180 μg·ml^?1 compared wi preparation, or with RNA extracted from the alkali-stable fraction of TMV suggest that apart from four legitimate virus-coded products of apparent M_r approx. 165 000, 110 000, 30 000 and 17 500 all other polypeptides arise from the overlapping 5′-proximal cistrons either by (i) site-selective endonucleolytic cleavage, (ii) sense codon misreading, or (iii) specific regions of secondary structure on TMV RNA which impede ribosome translocation.     

Endosomal proteolysis of insulin-like growth factor-I at its C-terminal D-domain by cathepsin B

IGF-I is degraded within the endosomal apparatus as a consequence of receptor-mediated endocytosis. However, the nature of the responsible protease and the position of the cleavage sites in the IGF-I molecule remain undefined. In vitro proteolysis of IGF-I using an endosomal lysate required an acidic pH and was sensitive to CA074, an inhibitor of the cathepsin B enzyme. By nondenaturing immunoprecipitation, the acidic IGF-I-degrading activity was attributed to the luminal species of endosomal cathepsin B with apparent molecular masses of 32- and 28-kDa. The cathepsin B precursor, procathepsin B, was processed in vitro within isolated endosomes at pH 5 or at 7 in the presence of ATP, the substrate of the vacuolar H(+)-ATPase. The rate of IGF-I hydrolysis using an endosomal lysate or pure cathepsin B was found to be optimal at pH 5-6 and moderate at pH 4 and 7. Competition studies revealed that EGF and IGF-I share a common binding site on the cathepsin B enzyme, with native IGF-I displaying the lowest affinity for the protease (IC50 approximately 1.5 microM). Hydrolysates of IGF-I generated at low pH by endosomal IGF-I-degrading activity and analyzed by reverse-phase HPLC and mass spectrometry revealed cleavage sites at Lys68-Ser69, Ala67-Lys68, Pro66-Ala67 and Lys65-Pro66 within the C-terminal D-domain of IGF-I. Treatment of human HepG2 hepatoma cells with the cathepsin B proinhibitor CA074-Me reduced, in vivo, the intracellular degradation of internalized [125I]IGF-I and, in vitro, the degradation of exogenous [125I]IGF-I incubated with the cell-lysates at pH 5. Inhibitors of cathepsin B and pro-cathepsin B processing, which abolish endosomal proteolysis of IGF-I and alter tumor cell growth and IGF-I receptor signalling, merit investigation as antimetastatic drugs.     

Tyrosine phosphorylation of a 94-kDa protein of human fibroblasts stimulated by streptococcal lipoteichoic acid

Lipoteichoic acid (LTA) is an amphipathic component of Gram-positive bacteria. Previous studies from this laboratory have shown that at low concentrations, ranging from 0.1 to 10.0 micrograms/ml, LTA binds to mammalian cells and stimulates mitogenic responses as demonstrated by increased DNA and RNA synthesis. Tyrosine kinase appears to be involved in the action of a number of mitogens including epidermal growth factor, platelet-derived growth factor, and insulin. In the present study, we report the novel finding that tyrosine protein kinase activity is increased in human fibroblasts treated with LTA. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of the whole cell lysate of fibroblasts cultured with 32Pi showed increased phosphorylation of a 94-kDa polypeptide. Alkali treatment of the gel resulted in a decreased intensity of the 94-kDa phosphorylated protein in control cells, but not in LTA-treated cells, suggesting the addition of phosphate groups to threonine or tyrosine residues. High voltage electrophoresis of the acid hydrolysate of the excised and eluted 94-kDa protein revealed that LTA stimulated the phosphorylation of tyrosine but not threonine residues. These results suggest that LTA acts on mammalian cells by phosphorylating tyrosine residues of certain proteins and thereby may regulate diverse functions of these cells.     

In vitro translation of the protease catalyzing Bombyx mori egg-specific protein and identification of a nascent peptide with biological activity

A yolk protein, egg-specific protein (ESP), of Bombyx mori is sequentially degraded by the ESP-specific protease which appears at the later stages of embryogenesis. In order to find the biological origin of this protease, an in vitro translation was done on RNAs prepared throughout embryogenesis using a rabbit reticulocyte lysate. Among several peptides translated, a 26-kDa peptide was selectively precipitated by the ESP protease antiserum. The mRNA activity increased slowly and then abruptly, reaching the maximum level on Day 8 of embryogenesis. By cotranslation with dog pancreatic microsomal membranes, the 26-kDa peptide was converted to a 24.5-kDa peptide, suggesting the cleavage of a signal peptide of 1.5 kDa. The direct incubation of the translation mixture with ESP failed to hydrolyze ESP, whereas the immunoprecipitate of the primary translation products clearly hydrolyzed ESP into the same peptides as were given by the authentic ESP protease. These results indicate that the protease becomes biologically active before chemical maturation.     

Maturation of giardiavirus capsid protein involves posttranslational proteolytic processing by a cysteine protease.

The double-stranded RNA genome of giardiavirus (GLV) has only two large open reading frame (ORFs). The 100-kDa capsid polypeptide (p100) is encoded by ORF1, whereas the only other viral polypeptide, the 190-kDa GLV RNA-dependent RNA polymerase (p190), is synthesized as an ORF1-ORF2 fusion protein by a (-1) ribosomal frameshifting. Edman degradation revealed that p100 was N-terminally blocked except for 2 to 5% of it that showed free N terminus starting from amino acid residue 33 of ORF1. Studies using antiserum targeted against amino acid residues 6 to 27 indicated that this region (NT) is absent from viral p100 and p190, while pulse-labelling experiments showed that NT is present in nascent p100 synthesized in GLV-infected Giardia lamblia but removed subsequently. In contrast, this region was retained in the two viral proteins synthesized in vitro, and it was not removed upon prolonged incubation or inclusion of microsomal fraction in the in vitro translation reaction mixtures. These results suggest that endoplasmic reticulum is not involved in the protein processing and that the precursors of p100 and p190 are incapable of cleaving themselves or each other. This specific cleavage was reproduced when lysates from GLV-infected G. lamblia were added, but not those from uninfected cells. The cleavage activity was relatively insensitive to phenylmethylsulfonyl fluoride, but it was inhibitable by leupeptin or E-64, two known specific inhibitors of cysteine protease. The possible origin of this processing activity is discussed.     

Cryptic chemotactic activity of fibronectin for human monocytes resides in the 120-kDa fibroblastic cell-binding fragment

Monocytes and lymphocytes form a second wave of infiltrating blood leukocytes in areas of tissue injury. The mechanisms for monocyte accumulation at these sites are not completely understood. Recently, however, fragments from extracellular matrix proteins including collagen, elastin, and fibronectin have been shown to induce monocyte chemotaxis. In this report we demonstrate that chemotactic activity for human monocytes is expressed when a 120-kDa fragment containing the RGDS cell-binding peptide is released from intact fibronectin or from larger fibronectin fragments. Monocytes, either from mononuclear cell Ficoll-Hypaque preparations (10-20% monocytes, 89-90% lymphocytes) or from elutriation preparations (95% monocytes, 5% lymphocytes), but not lymphocytes, migrated toward 120-kDa fragment preparations (10(-7) M) in blind-end chambers when the cells were separated from the chemoattractant by a 5-micron pore polycarbonate filter either alone or overlying a 0.45-micron pore nitrocellulose filter. Neutrophils migrated toward zymosan-activated serum but not toward 10(-5)-10(-8) M concentrations of the 120-kDa fragment. Intact fibronectin had no chemotactic activity for human monocytes. Fibronectin was isolated from citrated human plasma by sequential gelatin-Sepharose affinity and DEAE ion-exchange chromatography in the presence of buffers containing 1 mM phenylmethylsulfonyl fluoride to prevent fragmentation. Controlled enzymatic digestion with thermolysin cleaved fibronectin into 30 kDa fibrin, 45 kDa collagen, and 150/160-kDa cell and heparin domains. Upon prolonged digestion, purified 150/160-kDa fragments were cleaved into 120-kDa cell and 30/40-kDa heparin-binding fragments. Even though the intact fibronectin molecule, the 150/160-kDa fragments, and the 120-kDa fragment, have cell binding activity for Chinese hamster ovary fibroblasts, only the 120-kDa fragment expressed chemotactic activity for human monocytes. Thus, the 120-kDa fibroblastic cell-binding fragment contains a cryptic site for monocyte chemotaxis which is expressed upon enzymatic cleavage of fibronectin.     

Biosynthesis and intracellular processing of carbonic anhydrase in Chlamydomonas reinhardtii

Carbonic anhydrase (CA) of Chlamydomonas reinhardtii is a glycoprotein of 35 kDa which is localized outside the plasma membrane. The activity of CA was increased when the CO2 concentration during photoautotrophic growth was decreased to air level. After decreasing the CO2 concentration from 4% to 0.04%, several polypeptides including CA were induced continuously or transiently. To investigate the biosynthesis and intracellular processing of CA, the cells of wall-less mutant CW-15, which secretes CA into the culture medium, were pulse-labeled with radioactive arginine, chased, and radioactive proteins were immunoprecipitated with anti-CA serum. A 42-kDa polypeptide with isoelectric point (pI) of 7.1-7.3 was first synthesized. Within 5 min the molecular mass of this polypeptide was decreased to 35 kDa and it was then secreted into the culture medium within 30 min. This indicates that the former is the precursor form and the latter the mature form of CA. The primary translation product from poly(A)-rich RNA in a cell-free reticulocyte lysate system from a rabbit was a 38-kDa polypeptide. This was cotranslationally converted into the 42-kDa precursor in vitro in the presence of dog pancreatic microsomal membranes. As the 42-kDa precursor had a high affinity to concanavalin A, it was assumed to have a high-mannose-type oligosaccharide. The mature enzyme had a pI of 6.1-6.2 and was composed of more than two isoforms, which had a complex-type oligosaccharide with low affinity to concanavalin A. Chemical deglycosylation of the mature enzyme by trifluoromethanesulfonic acid indicated that the molecular mass of the polypeptide moiety was 32 kDa and the difference between this and the primary translation product suggests that cleavage of the polypeptide occurs during its biosynthesis.     

Activation of human furin precursor processing endoprotease occurs by an intramolecular autoproteolytic cleavage.

Human furin is a calcium-dependent serine endoprotease that can efficiently cleave many precursor proteins on the carboxyl side of the consensus cleavage sequence, -Arg-X-Lys/Arg-Arg-, both in vivo and in vitro. Analysis of furin proteins in extracts of cells infected with a vaccinia recombinant expressing human furin show that the enzyme is present as two prominent forms of 90 and 96 kDa. Because the structurally related bacterial subtilisins require endoproteolytic removal of the NH2-terminal pro-region by an autocatalytic intramolecular cleavage, we speculated that the size heterogeneity in the furin doublet similarly may result from a proteolytic removal of an NH2-terminal pro-region. Here we report identification of the 90-kDa furin NH2 terminus and, based on the reported sequence of the furin cDNA, demonstrate that this furin protein is derived from a larger precursor by an endoproteolytic cleavage on the COOH-terminal side of a consensus furin cleavage site, -Arg-Thr-Lys-Arg107-. Expression of mutant furin molecules containing an altered cleavage site (Arg104----Ala or Arg107----Gly) resulted in the production of only the 96-kDa furin protein. Assays of furin-dependent cleavage of a protein substrate in vitro showed that proteolytic activity was associated with the 90-kDa and not the 96-kDa furin protein, demonstrating that removal of the NH2-terminal pro-region is required for furin activity. Expression of a third furin construct containing a mutation of the active site aspartate (Asp153----Asn) similarly resulted in the expression of only the 96-kDa protein, suggesting that furin activation occurs by an autoproteolytic cleavage. Finally, the production of 90-kDa furin from either site-directed furin mutant could not be potentiated by overexpressing active furin, suggesting that the autoproteolytic activation was an intramolecular event.     

Removal of the genome-linked protein of foot-and-mouth disease virus by rabbit reticulocyte lysate.

Rabbit reticulocyte lysate cleaves the genome-linked protein VPg from foot-and-mouth disease virus (FMDV) RNA. This activity could be reliably monitored since removal of the protein resulted in a change in migration in polyacrylamide gels of the small specific 5' and fragment of the RNA (S fragment). The unlinking activity cleaved the bond between the tyrosine residue of VPg and the RNA to leave a 5' phosphate on the RNA. The 5' sequence of the RNA from which VPg had been removed by rabbit reticulocyte lysate was the same as that of FMDV mRNA isolated from infected cells. VPg released from the RNA was rapidly degraded by the rabbit reticulocyte lysate to material which eluted with the inclusion volume of a Sepharose 6B column and partitioned to the aqueous phase during phenol extraction. The unlinking activity was inhibited by heating the lysate to 56 degrees C, by sodium dodecyl sulfate (SDS), EDTA, and Zn2+ ions but was unaffected by reducing agents, a translation inhibitor, and a number of protease and RNase inhibitors.