PubMed Health⌕ Search

Biomedical subjects

J M Chirgwin

Publications and source records attributed to J M Chirgwin.

At least 19 recordsLinked to original sources
PubMed2000

Rabbit phosphoglucose isomerase/neuroleukin/autocrine motility factor: cloning via interspecies identity.

Phosphoglucose isomerase is the first committed enzyme of glycolysis. The protein also has a variety of biological activities on mammalian cells. The molecular basis of these extracellular functions is unclear, and the high resolution three-dimensional structure of a mammalian enzyme has not been described. We report here the cDNA and protein sequence for phosphoglucose isomerase from rabbit muscle. The sequence was obtained directly by PCR without the need to screen clones from a cDNA library and encoded active enzyme when expressed in bacterial cells. The 558 amino acid rabbit coding sequence is the same length as and highly similar (92% residue identity) to the sequences from human and pig and less so (88%) to the mouse enzyme. Non-conservative amino acid changes between the four mammalian sequences are concentrated in the first 35 and last five residues. The rabbit protein has an additional Cys residue and amino acid changes at five positions otherwise invariant in the mammalian enzymes.

Amino Acid Sequence↗
PubMed2000

Reproductive tissue renin gene expression in preeclampsia.

OBJECTIVE: Using an analogy with renin gene overexpression, low-renin hypertension animal models, we wished to test the hypothesis that renin gene expression is increased in decidua basalis in human gestation with preeclampsia. METHODS: Human placentas were obtained immediately after delivery from 11 control (C) and 11 preeclamptics (PE). Tissue samples were microdissected and renin gene expression in decidua basalis (DB), chorionic villi (CV), and decidua vera (DV) was measured using dot-blot hybridization. RESULTS: Overall renin gene expression is highest in decidua basalis (mean +/- SEM, 2.66 +/- 0.69 densitometry area units) compared to chorionic villi (mean +/- SEM, 1.85 +/- 0.5) or compared to decidua vera (mean +/- SEM, 1.63 +/- 0.9) (both t-tests p = 0.001 two-tailed and analysis of variance p = 0.0001). Renin gene expression in DB and in CV was similar in both preeclamptic and normal pregnancies (DB mean +/- SEM C 2.79 +/- 0.96 versus PE 2.54 +/- 1.04, and CV mean +/- SEM C 2.11 +/- 0.91 versus PE, 1.59 +/- 0. 44). Renin gene expression in DV was approximately threefold higher in tissues from preeclamptics compared to control (mean +/- SEM PE 2. 44 +/- 1.76 versus C 0.82 +/- 0.42). Using the median value of 0.5 units for DV as cutoff, the preeclamptics displayed higher renin gene expression (chi square p = 0.033, two tailed). CONCLUSION: Our data suggest that renin gene expression is increased in preeclampsia in decidua vera. This may explain previously reported increased renin secretion in uterine circulation in preeclampsia.

Adult↗
PubMed2000

Molecular mechanisms of tumor-bone interactions in osteolytic metastases.

In patients with advanced disease, several cancer types frequently metastasize to the skeleton, where they cause bone destruction. Osteolytic metastases are incurable and cause pain, hypercalcemia, fracture, and nerve compression syndromes. It was proposed over a century ago that certain cancers, such as that of the breast, preferentially metastasize to the favorable microenvironment provided by bone. Bone matrix is a rich store of immobilized growth factors that are released during bone resorption. Histological analysis of osteolytic bone metastases indicates that the bone destruction is mediated by the osteoclast rather than directly by the tumor cells. These observations suggest a vicious cycle driving the formation of osteolytic metastases: tumor cells secrete factors stimulating osteoclasts through adjacent bone marrow stromal cells; osteoclastic resorption in turn releases growth factors from the bone matrix; finally, locally released growth factors activate the tumor cells. This vicious cycle model has now been confirmed at the molecular level. In particular, transforming growth factor beta (TGF3beta) is abundant in bone matrix and released as a consequence of osteoclastic bone resorption. Bone-derived TGFbeta plays an integral role in promoting the development and progression of osteolytic bone metastases by inducing tumor production of parathyroid hormone-related protein (PTHrP), a known stimulator of osteoclastic bone resorption. In breast cancer cells TGFbeta appears to stimulate PTHrP secretion by a posttranscriptional mechanism through both Smad and p38 mitogen activated protein (MAP) kinase signaling pathways. Osteolytic metastases can be suppressed in vivo by inhibition of bone resorption, blockade of TGFbeta signaling in tumor cells, and by neutralization of PTHrP. Other factors released from bone matrix may also act on tumor cells in bone, which in turn may produce other factors that stimulate bone resorption, following the vicious cycle paradigm established for TGFbeta and PTHrP. An understanding at the molecular level of the mechanisms of osteolytic metastasis will result in more effective therapies for this devastating complication of cancer.

Bone Neoplasms↗
PubMed1999

NH2-terminal sequence truncation decreases the stability of bovine rhodanese, minimally perturbs its crystal structure, and enhances interaction with GroEL under native conditions.

The NH2-terminal sequence of rhodanese influences many of its properties, ranging from mitochondrial import to folding. Rhodanese truncated by >9 residues is degraded in Escherichia coli. Mutant enzymes with lesser truncations are recoverable and active, but they show altered active site reactivities (Trevino, R. J., Tsalkova, T., Dramer, G., Hardesty, B., Chirgwin, J. M., and Horowitz, P. M. (1998) J. Biol. Chem. 273, 27841-27847), suggesting that the NH2-terminal sequence stabilizes the overall structure. We tested aspects of the conformations of these shortened species. Intrinsic and probe fluorescence showed that truncation decreased stability and increased hydrophobic exposure, while near UV CD suggested altered tertiary structure. Under native conditions, truncated rhodanese bound to GroEL and was released and reactivated by adding ATP and GroES, suggesting equilibrium between native and non-native conformers. Furthermore, GroEL assisted folding of denatured mutants to the same extent as wild type, although at a reduced rate. X-ray crystallography showed that Delta1-7 crystallized isomorphously with wild type in polyethyleneglycol, and the structure was highly conserved. Thus, the missing NH2-terminal residues that contribute to global stability of the native structure in solution do not significantly alter contacts at the atomic level of the crystallized protein. The two-domain structure of rhodanese was not significantly altered by drastically different crystallization conditions or crystal packing suggesting rigidity of the native rhodanese domains and the stabilization of the interdomain interactions by the crystal environment. The results support a model in which loss of interactions near the rhodanese NH2 terminus does not distort the folded native structure but does facilitate the transition in solution to a molten globule state, which among other things, can interact with molecular chaperones.

Animals↗
PubMed1999

Properties of soluble fusions between mammalian aspartic proteinases and bacterial maltose-binding protein.

The mammalian aspartic proteinases procathepsin D and pepsinogen form insoluble inclusion bodies when expressed in bacteria. They become soluble but nonnative when synthesized as fusions to the carboxy terminus of E. coli maltose-binding protein (MBP). Since these nonnative states of the two aspartic proteinases showed no tendency to form insoluble aggregates, their biophysical properties were analyzed. The MBP portions were properly folded as shown by binding to amylose, but the aspartic proteinase moieties failed to bind pepstatin and lacked enzymatic activity, indicating that they were not correctly folded. When treated with proteinase K, only the MBP portion of the fusions was resistant to proteolysis. The fusion between MBP and cathepsin D had increased hydrophobic surface exposure compared to the two unfused partners, as determined by bis-ANS binding. Ultracentrifugal sedimentation analysis of MBP-procathepsin D and MBP-pepsinogen revealed species with very large and heterogeneous sedimentation values. Refolding of the fusions from 8 M urea generated proteins no larger than dimers. Refolded MBP-pepsinogen was proteolytically active, while only a few percent of renatured MBP-procathepsin D was obtained. The results suggest that MBP-aspartic proteinase fusions can provide a source of soluble but nonnative folding states of the mammalian polypeptides in the absence of aggregation.

ATP-Binding Cassette Transporters↗
PubMed1999

TGF-beta signaling blockade inhibits PTHrP secretion by breast cancer cells and bone metastases development.

Breast cancer frequently metastasizes to the skeleton, and the associated bone destruction is mediated by the osteoclast. Growth factors, including transforming growth factor-beta (TGF-beta), released from bone matrix by the action of osteoclasts, may foster metastatic growth. Because TGF-beta inhibits growth of epithelial cells, and carcinoma cells are often defective in TGF-beta responses, any role of TGF-beta in metastasis is likely to be mediated by effects on the surrounding normal tissue. However, we present evidence that TGF-beta promotes breast cancer metastasis by acting directly on the tumor cells. Expression of a dominant-negative mutant (TbetaRIIDeltacyt) of the TGF-beta type II receptor rendered the human breast cancer cell line MDA-MB-231 unresponsive to TGF-beta. In a murine model of bone metastases, expression of TbetaRIIDeltacyt by MDA-MB-231 resulted in less bone destruction, less tumor with fewer associated osteoclasts, and prolonged survival compared with controls. Reversal of the dominant-negative signaling blockade by expression of a constitutively active TGF-beta type I receptor in the breast cancer cells increased tumor production of parathyroid hormone-related protein (PTHrP), enhanced osteolytic bone metastasis, and decreased survival. Transfection of MDA-MB-231 cells that expressed the dominant-negative TbetaRIIDeltacyt with the cDNA for PTHrP resulted in constitutive tumor PTHrP production and accelerated bone metastases. These data demonstrate an important role for TGF-beta in the development of breast cancer metastasis to bone, via the TGF-beta receptor-mediated signaling pathway in tumor cells, and suggest that the bone destruction is mediated by PTHrP.

Animals↗
PubMed1998

Truncations at the NH2 terminus of rhodanese destabilize the enzyme and decrease its heterologous expression.

Rhodanese mutants containing sequential NH2-terminal deletions were constructed to test the distinct contributions of this region of the protein to expression, folding, and stability. The results indicate that the first 11 residues are nonessential for folding to the active conformation, but they are necessary for attaining an active, stable structure when expressed in Escherichia coli. Rhodanese species with up to 9 residues deleted were expressed and purified. Kinetic parameters for the mutants were similar to those of the full-length enzyme. Compared with shorter truncations, mutants missing 7 or 9 residues were (a) increasingly inactivated by urea denaturation, (b) more susceptible to inactivation by dithiothreitol, (c) less able to be reactivated, and (d) less rapidly inactivated by incubation at 37 degreesC. Immunoprecipitation showed that mutants lacking 10-23 NH2-terminal amino acids were expressed as inactive species of the expected size but were rapidly eliminated. Cell-free transcription/translation at 37 degreesC showed mutants deleted through residue 9 were enzymatically active, but they were inactive when deleted further, just as in vivo. However, at 30 degreesC in vitro, both Delta1-10 and Delta1-11 showed considerable activity. Truncations in the NH2 terminus affect the chemical stability of the distantly located active site. Residues Ser-11 through Gly-22, which form the NH2-proximal alpha-helix, contribute to folding to an active conformation, to resisting degradation during heterologous expression, and to chemical stability in vitro.

Amino Acid Sequence↗
PubMed1998

Order of fusions between bacterial and mammalian proteins can determine solubility in Escherichia coli.

We made fusions between Escherichia coli maltose-binding protein (MBP) and the mammalian aspartic proteinases pepsinogen or procathepsin D. When MBP was at the N-terminus, the fusions were soluble in E. coli. When the order was reversed, the chimeric proteins formed inclusion bodies. The data suggest that the solubility of fusion proteins is controlled by whether the protein domains emerging first from the ribosome normally fold into soluble or insoluble states. The soluble MBP-aspartic proteinase fusions were stable but proteolytically inactive. MBP-pepsinogen, however, was efficiently renatured from 8 M urea in vitro, suggesting that the E. coli cytoplasm does not support folding of the mammalian partner protein to the native state. Thus, inclusion body formation may be the consequence, rather than the cause, of non-native folding in vivo, and in E. coli soluble proteins may fold into states different from those reached in vitro.

ATP-Binding Cassette Transporters↗
PubMed1998

Solubility of proteins isolated from inclusion bodies is enhanced by fusion to maltose-binding protein or thioredoxin.

When the mammalian aspartic proteinases, procathepsin D or pepsinogen, are expressed in Escherichia coli both accumulate in inclusion bodies. While pepsinogen is efficiently refolded in vitro, recovery of procathepsin D is limited by insolubility. We expressed procathepsin D and pepsinogen in E. coli, with E. coli maltose-binding protein (MBP) or thioredoxin (trx) fused to their C-termini (aspartic proteinase-MBP or aspartic proteinase-trx). The fusion proteins were still found in inclusion bodies. However, the recovery of soluble procathepsin D-MBP and procathepsin D-trx after refolding was facilitated by the bacterial fusion partners. Maltose-binding protein was more efficient than thioredoxin in increasing the recovery of soluble protein. The vector, pET23bMBPH6, can be used for general expression of heterologous proteins in E. coli. The vector includes a histidine tag at the C-terminus of MBP to allow one-step purification of the fusion proteins under denaturing conditions. After purification, the protein of interest can be cleaved from MBP with factor Xa protease and separated from the MBP partner. Refolded pepsinogen-MBP and pepsinogen-trx were enzymatically active, but procathepsin D-MBP and procathepsin D-trx were soluble but largely inactive. The results show that the limited recovery of activity upon refolding of procathepsin D is not the consequence of competing aggregation. Thus, the fusions do not necessarily facilitate native refolding, but they do enhance the recovery of soluble protein. Such fusions could provide a system to study, in soluble form, folding states which are otherwise inaccessible because of aggregation and precipitation.

ATP-Binding Cassette Transporters↗
PubMed1998

Purification, amino acid sequence, and cDNA sequence of a novel calcium-precipitating proteolipid involved in calcification of corynebacterium matruchotii.

Corynebacterium matruchotii is a microbial inhabitant of the oral cavity associated with dental calculus formation. It produces membrane-associated proteolipid capable of inducing hydroxyapatite formation in vitro. This proteolipid was purified from chloroform:methanol extracts by chromatography on Sephadex LH-20 and migrated on SDS-polyacrylamide gel electrophoresis at 6-9 kDa. Removal of covalently attached acyl moieties by methanolic KOH decreased its molecular mass to approximately 5.5 kDa. The amino acid sequence of the apoproteolipid indicated a peptide of 50 amino acids, a calculated molecular weight of 5354 Da, and an isoelectric point of 4.28. Sequence analysis revealed an 8 amino acid sequence with homology to human phosphoprotein phosphatase 2A as well as several potential acylation sites and one phosphorylation site. The purified proteolipid induced calcium precipitation in vitro. Deacylation of the proteolipid by hydroxylamine treatment resulted in >50% loss of calcium-precipitating activity, suggesting that covalently attached lipids are required. Degenerate oligonucleotide primers, based on the amino acid sequence, were used to amplify the gene for the 5.5 kDa proteolipid from total chromosomal DNA of C. matruchotii by PCR. A 166 bp cDNA was isolated and sequenced, confirming the amino acid sequence of the proteolipid. Thus, we have sequenced a unique bacterial proteolipid that is involved in the formation of dental calculus by precipitating Ca2+ and possibly in transport of inorganic phosphate, necessary for hydroxyapatite formation.

Amino Acid Sequence↗
PubMed1997

Preformed GroES oligomers are not required as functional cochaperonins.

We have previously shown that the C-terminal sequence of GroES is required for oligomerization [Seale and Horowitz (1995), J. Biol. Chem. 270, 30268-30270]. In this report, we have generated a C-terminal deletion mutant of GroES with a significantly destabilized oligomer and have investigated its function in the chaperonin-assisted protein folding cycle. Removal of the two C-terminal residues of GroES results in a cochaperonin [GroESD(96-97)] that is monomeric at concentrations where GroES function is assessed. Using equilibrium ultracentrifugation, we measured the dissociation constant for the oligomer-monomer equilibrium to be 7.3 x 10(-34)M6. The GroESD(96-97) is fully active as a cochaperonin. This mutant is able to inhibit the ATPase activity of GroEL to levels comparable to wild-type GroES. It is also able to assist the refolding of urea-denatured rhodanese by GroEL. While GroESD(96-97) can function at levels comparable to wild-type GroES, higher concentrations of mutant are required to produce the same effect. These results support the idea that the performed GroES heptamer is not required for function, but they suggest that the oligomeric cochaperonin is most efficient.

Adenosine Triphosphatases↗
PubMed1995

The propeptide is nonessential for the expression of human cathepsin D.

When the 44-amino acid propeptide of human procathepsin D was deleted by mutagenesis in vitro, the mature protein was stably expressed and secreted from transfected mammalian cells. The secreted protein was correctly folded as judged by its binding to pepstatinylagarose. We were unable to detect lysosomal targeting of the propeptide-deleted protein, and targeting was not restored by the substitution of the propeptides from pepsin or renin. We conclude that its propeptide is not essential for the folding of nascent cathepsin D. Efficient lysosomal targeting in mammalian cells appears to require the precursor form of the molecule.

Animals↗
PubMed1995

Chinese hamster rhodanese cDNA: activity of the expressed protein is not blocked by a C-terminal extension.

We describe a cDNA from Chinese hamster ovary cells which encodes a protein 91 and 96% identical to bovine and rat mitochondrial rhodaneses, respectively. Recombinant protein was expressed from the cDNA in Escherichia coli, purified to homogeneity, and found to have kinetic properties nearly indistinguishable from those of the bovine enzyme, the only cloned rhodanese previously verified by characterization of the recombinant protein. The carboxyl-terminus of the enzyme is characterized by a duplicated tripeptide, which can be proteolytically processed in vivo. We constructed a mutant in which the last 5 amino acids were replaced by 28 residues of unrelated sequence. This protein was expressed, purified, and found to have kinetic constants similar to those of the wild-type enzyme. The functionally verified Chinese hamster rhodanese cDNA encodes a protein of 297 residues and differs from the rat enzyme at 13 positions. None of these substitutions occurs at residues suggested to play essential roles in catalysis or structural stabilization.

Animals↗
PubMed1995

Role of glycosylation in the expression of human procathepsin D.

Human procathepsin D carries two N-linked glycosylation sites at asparagine residues 70 and 199, widely separated on the surface of the folded protein. We created monoglycosylated procathepsin D molecules by site-directed mutagenesis in vitro of the individual glycosylation sites. With only two exceptions, all 12 mutants of this type were expressed efficiently in mammalian cells. The expressed proteins were stable, targeted to the lysosome, and partially secreted into the medium. When both glycosylation sites were eliminated, however, the expressed proteins (9 different mutants) were stable but most were not secreted and targeted poorly to the lysosome. Mammalian fibroblasts appear to sort nascent procathepsin D efficiently only if it is N-glycosylated. Procathepsin D monoglycosylated at N70 is readily distinguished from the endogenous protein in transfected human cells and thus provides an excellent substrate for studying lysosomal targeting in an homologous system.

Animals↗
PubMed1995

Lysine residues in the C-terminal lobe and lysosomal targeting of procathepsin D.

A major pathway to the lysosome for soluble hydrolases involves the 6-phosphorylation of mannose residues. The initial step in this reaction is catalyzed by a phosphotransferase which recognizes lysosomal precursors. We constructed mutants of human procathepsin D whose targeting to the lysosome could be assayed directly in intact cells. Eight lysine residues were individually converted to glutamic acid on the surface of the carboxyl terminal lobe of the protein. Mutants with as many as four Lys to Glu mutations were normally targeted to the lysosome and processed to the mature form of the enzyme in transfected cells. We conclude that the C-terminal lobe of procathepsin D may not carry a determinant essential for lysosomal targeting in intact fibroblasts.

Animals↗