Latent high molecular weight complex of transforming growth factor beta 1. Purification from human platelets and structural characterization

Human transforming growth factor beta 1 (TGF-beta 1) was purified as a latent high Mr complex from human platelets by a six-step procedure. Analysis by sodium dodecyl sulfate (SDS)-gel electrophoresis under reducing conditions revealed that the complex was composed of at least three components with apparent Mr values of 13,000, 40,000, and 125,000-160,000. The 13-kDa subunit was part of a disulfide-bonded dimer and was identified by amino acid sequencing as TGF-beta 1. The 40-kDa subunit was identified as the amino-terminal part of the TGF-beta 1 precursor lacking the hydrophobic signal sequence. Partial sequencing of the 125-160-kDa protein revealed that it is distinct from known proteins. The 40-kDa and the 125-160-kDa subunits are linked by disulfide bonds, forming a complex with an apparent Mr of 210,000 on SDS gels under nonreducing conditions. Experiments with partial reduction revealed that each complex contains two 40-kDa components linked by disulfide bonds; in addition, the dimer is disulfide-linked to one 125-160-kDa binding protein. TGF-beta 1 binds noncovalently to the 210-kDa complex, and in bound form, TGF-beta 1 is inactive. Incubations of the latent form of TGF-beta 1 at extreme pH values, in 0.02% SDS or in 8 M urea, lead to activation of TGF-beta 1, whereas the complex was resistant to treatment with 5 M NaCl or heat (3 min at 95 degrees C).     

Lefty proteins exhibit unique processing and activate the MAPK pathway

Lefty polypeptides, novel members of the transforming growth factor-beta (TGF-beta) superfamily, are involved in the formation of embryonic lateral patterning. Members of the TGF-beta superfamily require processing for their activation, suggesting cleavage to be an essential step for lefty activation. Transfection of different cell lines with lefty resulted in expression of a 42-kDa protein, which was proteolytically processed to release two polypeptides of 34 and 28 kDa. Since members of the proprotein convertase (PC) family cleave different TGF-beta factors and are involved in the establishment of embryonic laterality, we studied their role in lefty processing. Cotransfection analysis showed that PC5A processed the lefty precursor to the 34-kDa form in vivo, whereas furin, PACE4, PC5B, and PC7 had a limited activity. None of these PCs showed activity in the processing of the lefty polypeptide to the 28-kDa lefty form. The mutation of the consensus sequences for PC cleavage in the lefty protein allowed the lefty cleavage sites to be identified. Mutations of the sequence RGKR to GGKG (amino acids 74-77) and of RHGR to GHGR (amino acids 132-135) prevented the proteolytic processing of the lefty precursor to the 34- and 28-kDa forms, respectively. To identify the biologically active form of lefty, we studied the effect of lefty treatment on pluripotent P19 cells. Lefty did not induce Smad2 or Smad5 phosphorylation, Smad2/Smad4 heterodimerization, or nuclear translocation of Smad2 or Smad4, but activated the MAPK pathway in a time- and dose-dependent fashion. Further analysis showed the 28-kDa (but not the 34-kDa) polypeptide to induce MAPK activity. Surprisingly, the 42-kDa lefty protein was also capable of inducing MAPK activity, indicating that the lefty precursor is biologically active. The data support a molecular model of processing as a mechanism for regulation of lefty signaling.     

Synthesis and processing of the transmembrane envelope protein of equine infectious anemia virus.

The transmembrane (TM) envelope protein of lentiviruses, including equine infectious anemia virus (EIAV), is significantly larger than that of other retroviruses and may extend in the C-terminal direction 100 to 200 amino acids beyond the TM domain. This size difference suggests a lentivirus-specific function for the long C-terminal extension. We have investigated the synthesis and processing of the EIAV TM protein by immune precipitation and immunoblotting experiments, by using several envelope-specific peptide antisera. We show that the TM protein in EIAV particles is cleaved by proteolysis to an N-terminal glycosylated 32- to 35-kilodalton (kDa) segment and a C-terminal nonglycosylated 20-kDa segment. The 20-kDa fragment was isolated from virus fractionated by high-pressure liquid chromatography, and its N-terminal amino acid sequence was determined for 13 residues. Together with the known nucleotide sequence, this fixes the cleavage site at a His-Leu bond located 240 amino acids from the N terminus of the TM protein. Since the 32- to 35-kDa fragment and the 20-kDa fragment are not detectable in infected cells, we assume that cleavage occurs in the virus particle and that the viral protease may be responsible. We have also found that some cells producing a tissue-culture-adapted strain of EIAV synthesize a truncated envelope precursor polyprotein. The point of truncation differs slightly in the two cases we have observed but lies just downstream from the membrane-spanning domain, close to the cleavage point described above. In one case, virus producing the truncated envelope protein appeared to be much more infectious than virus producing the full-size protein, suggesting that host cell factors can select for virus on the basis of the C-terminal domain of the TM protein.     

Unique precursor structure of an extracellular protease, aqualysin I, with NH2- and COOH-terminal pro-sequences and its processing in Escherichia coli

Aqualysin I is a subtilisin-type serine protease which is secreted into the culture medium by Thermus aquaticus YT-1, an extremely thermophilic Gram-negative bacterium. The nucleotide sequence of the entire gene for aqualysin I was determined, and the deduced amino acid sequence suggests that aqualysin I is produced as a large precursor, consisting of at least three portions, an NH2-terminal pre-pro-sequence (127 amino acid residues), the protease (281 residues), and a COOH-terminal pro-sequence (105 residues). When the cloned gene was expressed in Escherichia coli cells, aqualysin I was not secreted. However, a precursor of aqualysin I lacking the NH2-terminal pre-pro-sequence (38-kDa protein) accumulated in the membrane fraction. On treatment of the membrane fraction at 65 degrees C, enzymatically active aqualysin I (28-kDa protein) was produced in the soluble fraction. When the active site Ser residue was replaced with Ala, cells expressing the mutant gene accumulated a 48-kDa protein in the outer membrane fraction. The 48-kDa protein lacked the NH2-terminal 14 amino acid residues of the precursor, and heat treatment did not cause any subsequent processing of this precursor. These results indicate that the NH2-terminal signal sequence is cleaved off by a signal peptidase of E. coli, and that the NH2- and COOH-terminal pro-sequences are removed through the proteolytic activity of aqualysin I itself, in that order. These findings indicate a unique four-domain structure for the aqualysin I precursor; the signal sequence, the NH2-terminal pro-sequence, mature aqualysin I, and the COOH-terminal pro-sequence, from the NH2 to the COOH terminus.     

Secretion of egg envelope protein ZPC after C-terminal proteolytic processing in quail granulosa cells.

In avian species, an egg envelope homologous to the mammalian zona pellucida is called the perivitelline membrane. We have previously reported that one of its components, a glycoprotein homologous to mammalian ZPC, is synthesized in the granulosa cells of the quail ovary. In the present study, we investigated the proteolytic cleavage of the newly synthesized ZPC and the secretion of ZPC from the granulosa cells. Western blot analysis of the cell lysates demonstrated that the 43-kDa protein is the precursor of mature ZPC (proZPC), and is converted to the 35-kDa protein before secretion. The accumulation of proZPC in the presence of brefeldin A, and conversion of proZPC to ZPC in the presence of monensin, indicate the possibility that the proteolytic processing of ZPC occurs in the Golgi apparatus. An analysis of amino-acid sequence identified that the C terminus of mature ZPC protein is Phe360, and the N-terminal amino-acid sequence of the proZPC-derived fragment was determined as Asp363. These results suggest that newly synthesized ZPC is cleaved at the consensus furin cleavage site, and the resulting two basic residues at the C terminus are subsequently trimmed off to generate mature ZPC prior to secretion.     

Regulation of biological activity and matrix assembly of laminin-5 by COOH-terminal, LG4-5 domain of alpha3 chain

The basement membrane protein laminin-5 (LN5; alpha3beta3gamma2) undergoes specific proteolytic processing of the 190-kDa alpha3 chain to the 160-kDa form after the secretion, releasing its COOH-terminal, LG4-5 domain. To clarify the biological significance of this processing, we tried to express a recombinant precursor LN5 with a 190-kDa alpha3 chain (pre-LN5), in which the cleavage sequence Gln-Asp was changed to Ala-Ala by point mutation. When the wild-type and mutated LN5 heterotrimers were expressed in HEK293 cells, the wild-type alpha3 chain was completely cleaved, whereas the mutated alpha3 chain was partially cleaved at the same cleavage site (Ala-Ala). pre-LN5 was preferentially deposited on the extracellular matrix, but this deposition was effectively blocked by exogenous heparin. This suggests that interaction between the LG4-5 domain and heparan sulfate proteoglycans on the cell surface and/or extracellular matrix is important in the matrix assembly of LN5. Next, we purified both pre-LN5 and the mature LN5 with the processed, 160-kDa alpha3 chain (mat-LN5) from the conditioned medium of the HEK293 cells and compared their biological activities. mat-LN5 showed higher activities to promote cell adhesion, cell scattering, cell migration, and neurite outgrowth than pre-LN5. These results indicate that the proteolytic removal of LG4-5 from the 190-kDa alpha3 chain converts the precursor LN5 from a less active form to a fully active form. Furthermore, the released LG4-5 fragment stimulated the neurite outgrowth in the presence of mat-LN5, suggesting that LG4-5 synergistically enhances integrin signaling as it is released from the precursor LN5.     

A 15-kDa interferon-induced protein is derived by COOH-terminal processing of a 17-kDa precursor

An interferon-induced 15-kDa protein is synthesized from a precursor of higher molecular weight; the precursor contains 165 amino acids (17 kDa), whereas the stable product (15 kDa) contains 156 amino acids. The stable 15-kDa form is derived from the precursor 17-kDa form by the removal of eight amino acids from the COOH terminus and the methionine from the NH2 terminus. The existence of the precursor 17-kDa protein can be demonstrated after brief periods of in vivo labeling with [35S]methionine and by translation of mRNA in vitro.     

The pro domain of pre-pro-transforming growth factor beta 1 when independently expressed is a functional binding protein for the mature growth factor

Transforming growth factor beta 1 (TGF-beta 1) is proteolytically derived from the carboxyl terminus of a 390 amino acid precursor molecule termed pre-pro-TGF-beta 1. Previous studies have suggested that the pro piece of pre-pro-TGF-beta 1 may play an important role in the formation of an inactive, latent complex. These latent forms are thought to be important in the regulation of TGF-beta 1 activity. To understand this latent complex in more detail, we have expressed the pro domain of pre-pro-TGF-beta 1 in tissue culture cells independent of the mature growth factor. A stop codon was genetically engineered into the cDNA of pre-pro-TGF-beta 1 by changing the Arg-278 codon from CGA to the STOP codon TGA. The resulting protein is truncated just prior to the amino-terminal Ala residue of the mature growth factor. Transient expression studies and immunoblotting indicate that this pro piece is readily made and secreted by the COS-1 cells; the major form of the expressed pro piece, when analyzed by SDS-polyacrylamide gel electrophoresis, behaves as a disulfide-linked dimer (Mr 80,000). Bioassays, using mink lung indicator cells, reveal that the pro domain forms an inactive complex with exogenously added mature TGF-beta 1. Treatment of this complex with heat or acid results in the release of active TGF-beta 1, indicating an in vitro structure similar to natural, latent TGF-beta 1 complexes. The pro piece from TGF-beta 1 was also found to form latent structures with two closely related family members, TGF-beta 1.2 and TGF-beta 2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure of yeast pGKL 128-kDa killer-toxin secretion signal sequence. Processing of the 128-kDa killer-toxin-secretion-signal-alpha-amylase fusion protein.

The linear double-stranded DNA plasmid pGKL1 in yeast encodes a killer toxin consisting of 97-kDa, 31-kDa and 28-kDa subunits. A 128-kDa protein precursor of the 97-kDa and 31-kDa subunits, was first synthesized with a 29-amino-acid extension at its NH2-terminus as a secretion signal sequence. In the present study, the property of this signal sequence was studied by the analysis of a fusion protein with mouse alpha-amylase. Using the secretion signal sequence of the killer protein, the mouse alpha-amylase was successfully secreted into the culture medium. An intracellular precursor form of alpha-amylase was identified and purified. Analysis of the NH2-terminal sequence of this precursor molecule indicated that it corresponded to the secretory intermediate (pro form) of alpha-amylase with the removal of the hydrophobic segment (Met1-Gly16) of the secretion signal. Both the secretion of alpha-amylase into the culture medium and the detection of the pro-alpha-amylase species in the cells were prohibited by a sec 11 mutation, or by the conversion of Gly to Val at the 16th position of the secretion signal. These results strongly suggest that the cleavage occurs between Gly16 and Leu17 by a signal peptidase, and that this cleavage is required for the secretion of alpha-amylase into the medium. Based on the data from the NH2-terminal amino acid sequences of secreted alpha-amylases, we conclude that the 29-amino-acid secretion signal present in the 128-kDa killer toxin precursor protein is a prepro structure.     

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.     

Structure of the cel-3 gene from Fibrobacter succinogenes S85 and characteristics of the encoded gene product, endoglucanase 3.

The cel-3 gene cloned from Fibrobacter succinogenes into Escherichia coli coded for the enzyme EG3, which exhibited both endoglucanase and cellobiosidase activities. The gene had an open reading frame of 1,974 base pairs, coding for a protein of 73.4 kilodaltons (kDa). However, the enzyme purified from the osmotic shock fluid of E. coli was 43 kDa. The amino terminus of the 43-kDa protein matched amino acid residue 266 of the protein coded for by the open reading frame, indicating proteolysis in E. coli. In addition to the 43-kDa protein, Western immunoblotting revealed a 94-kDa membranous form of the enzyme in E. coli and a single protein of 118 kDa in F. succinogenes. Thus, the purified protein appears to be a proteolytic degradation product of a native protein which was 94 kDa in E. coli and 118 kDa in F. succinogenes. The discrepancy between the molecular weight expected on the basis of the DNA sequence and the in vivo form may be due to anomalous migration during electrophoresis, to glycosylation of the native enzyme, or to fatty acyl substitution at the N terminus. One of two putative signal peptide cleavage sites bore a strong resemblance to known lipoprotein leader sequences. The purified 43-kDa peptide exhibited a high Km (53 mg/ml) for carboxymethyl cellulose but a low Km (3 to 4 mg/ml) for lichenan and barley beta-glucan. The enzyme hydrolyzed amorphous cellulose, and cellobiose and cellotriose were the major products of hydrolysis. Cellotriose, but not cellobiose, was cleaved by the enzyme. EG3 exhibited significant amino acid sequence homology with endoglucanase CelC from Clostridium thermocellum, and as with both CelA and CelC of C. thermocellum, it had a putative active site which could be aligned with the active site of hen egg white lysozyme at the highly conserved amino acid residues Asn-44 and Asp-52.     

Expression and analysis of the human cytomegalovirus UL80-encoded protease: identification of autoproteolytic sites.

The 45-kDa assembly protein of human cytomegalovirus is encoded by the C-terminal portion of the UL80 open reading frame (ORF). For herpes simplex virus, packaging of DNA is accompanied by cleavage of its assembly protein precursor at a site near its C terminus, by a protease encoded by the N-terminal region of the same ORF (F. Liu and B. Roizman, J. Virol. 65:5149-5156, 1991). By analogy with herpes simplex virus, we investigated whether a protease is contained within the N-terminal portion of the human cytomegalovirus UL80 ORF. The entire UL80 ORF was expressed in Escherichia coli, under the control of the phage T7 promoter. UL80 should encode a protein of 85 kDa. Instead, the wild-type construct produces a set of proteins with molecular masses of 50, 30, 16, 13, and 5 kDa. In contrast, when mutant UL80 is deleted of the first 14 amino acids, it produces only an 85-kDa protein. These results suggest that the UL80 polyprotein undergoes autoproteolysis. We demonstrate by deletional analysis and by N-terminal sequencing that the 30-kDa protein is the protease and that it originates from the N terminus of UL80. The UL80 polyprotein is cleaved at the following three sites: (i) at the C terminus of the assembly protein domain, (ii) between the 30- and 50-kDa proteins, and (iii) within the 30-kDa protease itself, which yields the 16- and 13-kDa proteins and may be a mechanism to inactivate the protease.     

Phosphorylation of intracellular precursors of human IL-1

The human IL-1 molecules (IL-1 alpha and IL-1 beta) are post-translationally cleaved from 31-kDa precursor to 18-kDa biologically active molecules. During the course of studies of post-translational modifications of human IL-1, we have observed that although LPS induced the production of both intracellular IL-1 alpha and IL-1 beta in human monocytes, [32P]orthophosphate labeling of these cells revealed that intracellular precursor of IL-1 alpha (pre-IL-1 alpha) to be phosphorylated at least 10-fold more than intracellular pre-IL-1 beta. However, no 32P-incorporation could be detected in the 18-kDa processed IL-1 alpha and IL-1 beta. Analysis by TLC revealed that the major phosphorylation site occurred at serine residue(s). The 32P was incorporated into multiply cleaved precursors of IL-1 alpha, which appeared in the absence of protease inhibitors. Since the smallest Mr pre-IL-1 alpha that was labeled with 32P was 22 kDa, the phosphorylated serine residue is presumably located adjacent to a sequence of four basic amino acids located in the 4-kDa region at the amino terminus of the 22-kDa precursor of IL-1 alpha. This serine residue might also be a major phosphorylation site for a cAMP-dependent protein kinase. This hypothesis was substantiated by the demonstration that a synthetic peptide analogue of this region (residue 84 to 112) could be similarly phosphorylated in vitro by a cAMP-dependent protein kinase. Furthermore, a truncated pre-IL-1 alpha (residue 64 to 271) and a "fusion" protein containing staphylococcal protein A and an amino-terminal half-portion of pre-IL-1 alpha (residue 1 to 112), but not mature IL-1 alpha (residue 113 to 271), could also be phosphorylated by cAMP-dependent protein kinase. There is no comparable amino acid sequence in IL-1 beta which could be expected to be phosphorylated by a cAMP-dependent protein kinase. The physiologic relevance of phosphorylation of pre-IL-1 alpha was investigated. The data showed that phosphorylation of truncated pre-IL-1 alpha greatly enhanced its susceptibility to digestion by trypsin and promoted the conversion of pre-IL-1 alpha to the more biologically active IL-1. Although the precise role of the rather selective phosphorylation of pre-IL-1 alpha is not known, our findings do suggest that the phosphorylation of serine close to dibasic/tetrabasic amino acid sequence functions to facilitate the processing and/or release of IL-1 alpha.     

Caspase-dependent N-terminal cleavage of influenza virus nucleocapsid protein in infected cells

The nucleocapsid protein (NP) (56 kDa) of human influenza A viruses is cleaved in infected cells into a 53-kDa form. Likewise, influenza B virus NP (64 kDa) is cleaved into a 55-kDa protein with a 62-kDa intermediate (O. P. Zhirnov and A. G. Bukrinskaya, Virology 109:174-179, 1981). We show now that an antibody specific for the N terminus of influenza A virus NP reacted with the uncleaved 56-kDa form but not with the truncated NP53 form, indicating the removal of a 3-kDa peptide from the N terminus. Amino acid sequencing revealed the cleavage sites ETD16*G for A/Aichi/68 NP and sites DID7*G and EAD61*V for B/Hong Kong/72 NP. With D at position -1, acidic amino acids at position -3, and aliphatic ones at positions -2 and +1, the NP cleavage sites show a recognition motif typical for caspases, key enzymes of apoptosis. These caspase cleavage sites demonstrated evolutionary stability and were retained in NPs of all human influenza A and B viruses. NP of avian influenza viruses, which is not cleaved in infected cells, contains G instead of D at position 16. Oligopeptide DEVD derivatives, specific caspase inhibitors, were shown to prevent the intracellular cleavage of NP. All three events, the NP cleavage, the increase of caspase activity, and the development of apoptosis, coincide in cells infected with human influenza A and B viruses. The data suggest that intracellular cleavage of NP is exerted by host caspases and is associated with the development of apoptosis at the late stages of infection.     

Expression, purification, and characterization of a novel methyl parathion hydrolase

The mpd gene coding for a novel methyl parathion hydrolase (MPH) was previously reported and its putative open reading frame was also identified. To further confirm its coding region, the intact region encoding MPH was obtained by PCR and expressed in Escherichia coli as a hexa-His C-terminal fusion protein. The fusion protein was purified to homogeneity by metal-affinity chromatography. The enzyme activity and zymogram assay showed that the fusion protein was functional in degrading methyl parathion. The amino terminal sequencing of the purified recombinant MPH indicated that a signal peptide of the first 35 amino acids was cleaved from its precursor to form active MPH. A rat polyclonal antiserum was raised against the purified mature fusion protein. The results of Western blot and zymogram demonstrated that mature MPH in native Plesiomonas sp. strain M6 was also processed from its precursor by cleavage of a putative signal peptide at the amino terminus. The production of active MPH in E. coli was greatly improved after the coding region for the signal peptide was deleted. HPLC gel filtration of the purified mature recombinant MPH revealed that the MPH was a monomer.     

Identification of a novel transforming growth factor-beta (TGF-beta 5) mRNA in Xenopus laevis

A novel transforming growth factor-beta (TGF-beta) mRNA of about 3.0 kilobases, which encodes a putative protein of 382 amino acids, has been identified in amphibians by cDNA cloning. This mRNA, which we designate as TGF-beta 5, is developmentally regulated and highly expressed beginning at early neurula (stage 14) and in many adult tissues in Xenopus laevis. Following the first methionine, the putative precursor protein has a hydrophobic region, approximately 22 amino acids long, which probably represents a signal sequence, similar to that found in TGF-beta s 1-3. The precursor also has potential sites for glycosylation, integrin binding (RGD), and a tetrabasic amino acid (RKKR) site for potential cleavage of the precursor peptide to a biologically active protein. The putative mature protein consists of 112 amino acids with 9 cysteines and has 76, 66, 69, and 72% identity to TGF-beta s 1-4, respectively.     

Site-directed mutagenesis of cysteine residues in the pro region of the transforming growth factor beta 1 precursor. Expression and characterization of mutant proteins

Three cysteine residues are located in the pro region of the transforming growth factor beta 1 (TGF-beta 1) precursor at amino acid positions 33, 223, and 225. Previous studies (Gentry, L. E., Lioubin, M. N., Purchio, A. F., and Marquardt, H. (1988) Mol. Cell. Biol. 8, 4162-4168) with purified recombinant TGF-beta 1 (rTGF-beta 1) precursor produced by Chinese hamster ovary (CHO) cells revealed that Cys-33 can form a disulfide bond with at least 1 cysteine residue in mature TGF-beta 1, contributing to the formation of a 90-110-kDa protein. We now show that Cys-223 and Cys-225 form interchain disulfide bonds. Site-directed mutagenesis was used to change these Cys codons to Ser codons, and mutant constructs were transfected into COS cells. Analysis of recombinant proteins by immunoblotting showed that by substituting Cys-33 the 90-110-kDa protein is not formed, and thus, more mature dimer (24 kDa) is obtained, corresponding to a 3- to 5-fold increase in biological activity. Substitution of Cys-223 and/or Cys-225 resulted in near wild-type levels of mature TGF-beta 1. Furthermore, cells transfected with plasmid coding for Ser at positions 223 and 225 expressed only monomeric precursor proteins and released bioactive TGF-beta 1 that did not require acid activation, suggesting that dimerization of the precursor pro region may be necessary for latency.     

Transforming growth factor beta 1 induces apoptosis through cleavage of BAD in a Smad3-dependent mechanism in FaO hepatoma cells

Transforming growth factor beta (TGF-beta) induces apoptosis in a variety of cells. We have previously shown that TGF-beta 1 rapidly induces apoptosis in the FaO rat hepatoma cell line. We have now studied the effect of TGF-beta 1 on the expression of different members of the Bcl-2 family in these cells. We observed no detectable changes in the steady-state levels of Bcl-2, Bcl-X(L), and Bax. However, TGF-beta 1 induced caspase-dependent cleavage of BAD at its N terminus to generate a 15-kDa truncated protein. Overexpression of the 15-kDa truncated BAD protein enhanced TGF-beta 1-induced apoptosis, whereas a mutant BAD resistant to caspase 3 cleavage blocked TGF-beta 1-induced apoptosis. Overexpression of Smad3 dramatically enhanced TGF-beta 1-induced cleavage of BAD and apoptosis, whereas antisense Smad3 blocked TGF-beta 1-induced apoptosis and BAD cleavage. These results suggest that TGF-beta 1 induces apoptosis through the cleavage of BAD in a Smad3-dependent mechanism.     

Differential Cleavage of Provasopressin by the Major Molecular Forms of SPC3

Abstract: We have investigated the roles of full-length and carboxyl-terminus-truncated forms of the subtilisin-like prohormone convertase SPC3 in the processing of the radiolabeled vasopressin and oxytocin precursors, in vitro. We found SPC3 cleaves provasopressin at both the vasopressin-neurophysin and neurophysin-glycopeptide processing sites. Prooxytocin is cleaved by SPC3 at the oxytocin-neurophysin cleavage site. However, our results reveal differences in processing of provasopressin by the different molecular forms of SPC3. In incubations where the rate of autocatalytic carboxyl-terminus truncation of SPC3 was dramatically reduced, 86-kDa SPC3, which has an unprocessed carboxyl terminus, cleaved provasopressin at the neurophysin-glycopeptide junction. Cleavage at the vasopressin-neurophysin junction only occurred with the appearance of carboxyl-terminus-truncated forms of the enzyme. Incubations containing 64-kDa SPC3 or 64-kDa SPC3-T, a recombinant form of SPC3 truncated 14 amino acids beyond the conserved carboxyl-terminal "P-domain," rapidly cleaved provasopressin at both the vasopressin-neurophysin and neurophysin-glycopeptide junctions. Our results also suggest that prooxytocin is unable to be cleaved by the 86-kDa form of SPC3. We propose that SPC3 should be considered as a candidate endoprotease in the biosynthesis of vasopressin. Furthermore, we suggest that the carboxyl terminus of SPC3 alters the cleavage specificity of SPC3.