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Biomedical subjects

M C Beinfeld

Publications and source records attributed to M C Beinfeld.

At least 19 recordsLinked to original sources
PubMed2000

PC2 and 7B2 null mice demonstrate that PC2 is essential for normal pro-CCK processing.

Analysis of CCK content in extracts of whole forebrain from PC2 and 7B2 null mouse brain showed a significant decrease relative to wild-type brains. More detailed analysis revealed that CCK 8 amide levels in cerebral cortex and forebrain regions were more decreased than in hypothalamus. CCK 8 content in PC2 null mouse intestines was identical to control. Null mutant brains contained less CCK 8 than wild type and no other forms were seen when analyzed by gel filtration chromatography. No brain area examined was completely devoid of CCK, suggesting that other enzymes can partially compensate for the loss of PC2. This is the first demonstration that any endoprotease is important for CCK processing but also suggest the presence of a redundant system to ensure production of active CCK in the brain.

Animals↗
PubMed2000

Lovastatin is a potent inhibitor of cholecystokinin secretion in endocrine tumor cells in culture.

Lovastatin prevents isoprene synthesis thereby affecting the structural organization of proteins involved in protein transport and secretion. Lovastatin at 1 microM decreases CCK 8 secretion by over 50% in WE cells and in CCK 8 expressing AtT20 cells. At 10 microM CCK 8 secretion was inhibited by two thirds and at 100 microM, cytotoxic effects were observed in both cell types. Addition of mevalonate does not restore CCK secretion and stimulation of secretion by forskolin is also partially inhibited. Cellular content of CCK 8 and pro-CCK were not altered in either of these cell lines except at 100 microM lovastatin. Our results clearly demonstrate that lovastatin at 1 microM strongly inhibits CCK 8 secretion at multiple levels while having little or no effect on its synthesis. This effect on secretion may be partly responsible for the adverse gastrointestinal side effects of lovastatin in patients.

Animals↗
PubMed1998

Prohormone convertase 1 (PC1) when expressed with pro cholecystokinin (pro CCK) in L cells performs three endoproteolytic cleavages which are observed in rat brain and in CCK-expressing endocrine cells in culture, including the production of glycine and arginine extended CCK8.

Pro CCK was expressed in an L cell line engineered to express PC1 and the products secreted into the media were characterized by a combination of RIA, gel filtration and HPLC. PC1 released from L cells, cleaved pro CCK generating the amino terminal pro peptide. PC1 also generated a peptide which after carboxypeptidase B treatment, was detected with an antiserum specific for CCK Gly. Neither of these peptides was found in media from L cells expressing pro CCK alone. This CCK Gly immunoreactive peptide was similar in size to CCK 8, and after treatment with arylsulfatase and carboxypeptidase B, it co-eluted on HPLC with unsulfated CCK 8 Gly. These results agree with previous studies which support a role for PC1 in generation of CCK 8. This is the first demonstration that PC1 acting alone is able to cleave pro CCK liberating the amino terminal pro peptide and a glycine and arginine extended CCK 8 which is the immediate precursor of CCK 8 amide.

Amino Acid Sequence↗
PubMed1998

Reduced levels of substance P in the brains of Cpe(fat)/Cpe(fat) mice.

This study examines the role of carboxypeptidase E (CPE) in processing pro tachykinin to form the final bioactive amidated undecapeptide, substance P (SP) in various rat brain regions. Cpe(fat)/Cpe(fat) mice brain tissue was analyzed for total SP forms (including intermediates), and final amidated SP was compared to Cpe+/Cpe+ and Cpe+/Cpe- controls. In all brain regions tested by radioimmunoassay, amidated fully processed SP was more than fivefold lower in Cpe(fat)/Cpe(fat) mice than in controls whereas total SP species levels were unchanged. This demonstrates that CPE is required for normal SP proteolytic processing. Substance P has numerous functions in the brain; therefore, SP deficiency due to the CPE mutation may contribute to the obese phenotype or even to other phenotypes not yet described in Cpe(fat)/Cpe(fat) mice.

Animals↗
PubMed1998

Identification of glycine-extended CCK peptides in endocrine cells and modulation of CCK amide and CCK Gly content and secretion from endocrine tumor cells by an inhibitor of amidation.

Immunoreactive glycine-extended CCK peptides are found in normal mouse cerebral cortex and are very abundant in some CCK expressing endocrine tumor cells in culture. The glycine-extended forms in mouse cortex and in cell lines mirror their respective amidated forms. Mouse cerebral cortex, mouse AtT20 and rat WE cells produce mainly CCK 8 amide and CCK 8 Gly. In contrast, mouse intestinal STC-1 cells produce CCK 22 and CCK 8 amide along with forms of CCK Gly which are slightly larger than their respective amidated forms. The CCK 8 Gly-like peptide from AtT20 cells, after desulfation, co-eluted on HPLC with unsulfated CCK 8 Gly. Addition of copper and ascorbate to culture medium of WE cells caused a small increase in secretion of amidated CCK, without changing cellular levels of this peptide. Treatment with the amidation inhibitor diethyldithiocarbamate greatly decreased cellular content and secretion of CCK amide while it increased cellular content and secretion of CCK Gly. These results provide further evidence that glycine-extended CCK peptides are the immediate precursors of amidated CCK peptides.

Amides↗
PubMed1998

Prohormone and proneuropeptide processing. Recent progress and future challenges.

Our knowledge of prohormone and proneuropeptide processing and its relationship to the secretory pathway has advanced significantly in the last five years. The recent discovery of the prohormone convertase family of proteolytic enzymes has provided new candidates for the prohormone and proneuropeptide convertases. The increasing appreciation of the role of proteolysis in diverse cellular processes has also brought the prohormone processing field closer to the fields of growth factor processing, the role of host proteases in viral and bacterial pathogenesis and toxicity, control of the cell cycle, inflammation, and apoptosis. The last five years have been very productive, but the most interesting questions are still unanswered. Which enzymes are actually responsible for prohormone cleavages in specific tissues? What structural features of the prohormones determine where it will be processed or how it is recognized as secretory material by the sorting machinery? How is tissue-specific processing determined and regulated? The availability of protease knockout mice and and a more detailed understanding of the complex biosynthetic activation of these enzymes will provide at least some of the answers.

Animals↗
PubMed1998

Adult carboxypeptidase E-deficient fat/fat mice have a near-total depletion of brain CCK 8 accompanied by a massive accumulation of glycine and arginine extended CCK: identification of CCK 8 Gly as the immediate precursor of CCK 8 in rodent brain.

Cholecystokinin (CCK) amide concentrations were reduced over 85% in all the major brain regions of carboxypeptidase E (Cpe)(fat)/Cpe(fat) mice in comparison to control mice. Using an radioimmunoassay (RIA) specific for glycine-extended CCK (CCK Gly), low levels of CCK Gly were detected in control (0.65 ng/g tissue) and were even lower in Cpe(fat)/Cpe(fat) (0.246 ng/g) mice brain extracts. After treatment with carboxypeptidase B, the level of CCK Gly in Cpe(fat)/Cpe(fat) in these brain extracts was elevated to 33.5 ng/g, about 51-fold higher than in control. On gel-filtration chromatography and high-performance liquid chromatography (HPLC), this material coeluted with CCK 8 Gly. These results demonstrate that CPE is required for the correct processing of arginine- and glycine-extended CCK in all major regions of the mouse brain. These results support the hypothesis that CCK 8 Gly is the immediate precursor of CCK 8 amide in mouse brain, not larger amidated forms like CCK 22 or CCK 33.

Animals↗
PubMed1997

Prohormone convertase 1 is necessary for the formation of cholecystokinin 8 in Rin5F and STC-1 cells.

Several immortalized cell lines serve as models for procholecystokinin (pro-CCK) processing. Rin5F cells, derived from a rat insulinoma, and STC-1 cells, derived from a murine intestinal tumor, process pro-CCK mainly to amidated CCK 8. Both also make significant quantities of amidated CCK 22, a slightly larger form found in the gut. Many modifications are necessary during pro-CCK processing including cleavages performed by endoproteases, the identities of which are unknown. A candidate endoprotease is prohormone convertase 1 (PC1) also known as PC3, a Ca2+-dependent serine endoprotease of the subtilisin family. Constitutive expression of antisense PC1 message in stably transfected Rin5F cells resulted in a significant reduction of the cellular content of CCK 8 as measured by radioimmunoassay. Several affected cell lines displayed about 80% reduction in CCK content in early passages after transfection. Expression of antisense PC1 message in these cell lines resulted in a selective depletion of CCK 8 and a comparative sparing of CCK 22. The induction of antisense PC1 message within a single subclone of Rin5F cells using the Lac Switch system also resulted in a significant inhibition of CCK content. Expression of antisense PC1 message in a stably transfected STC-1 cell line also resulted in a decrease in CCK content and in PC1 protein expression, and the specific depletion of CCK 8 with comparative sparing of CCK 22. These observations support the hypothesis that PC1 is necessary for pro-CCK processing in Rin5F and STC-1 cells and suggests a role for PC1 endoprotease in the biosynthesis of CCK 8 in vivo.

Animals↗
PubMed1997

Cleavage of CCK 33 by recombinant PC2 in vitro.

The specificity and kinetic properties of CCK 33 cleavage by recombinant prohormone convertase 2 (PC2) was investigated using a combination of Sephadex G-50 chromatography, HLPC and RIA methods. It is shown that CCK 33 can be cleaved effectively by PC2 to form CCK 8, the reaction of which has a K(m) of 104.8 microM. No CCK 22 or other products were detected and the reaction is completely inhibited by the PC2 inhibitor, p-CMS (p-chloromercuriphenylsulfonic acid), suggesting that the cleavage is PC2 specific. The time course of this reaction shows an initial lag phase followed by a rapid increase in velocity consistent with a previously reported spontaneous transformation from a 71 kDa relatively inactive form into a more active form of 62 kDa (1). PC2 did not cleave recombinant pro CCK or CCK 1-21. These results demonstrate that PC2, an enzyme usually associated with dibasic cleavages, can cleave easily at single basic residues.

4-Chloromercuribenzenesulfonate↗
PubMed1997

CCK biosynthesis and processing: recent progress and future challenges.

The peptide cholecystokinin (CCK), like other peptides which pass through the regulated secretory pathway, undergoes a number of post-translational modifications during its biosynthesis including tyrosine sulfation, endoproteolytic cleavage, and trimming by carboxypeptidases. This minireview summarizes what is known about this process in endocrine cells and in the Cpe(fat)/Cpe(fat) mouse and points out what challenges remain for future research.

Animals↗
PubMed1997

Expression of rat pro cholecystokinin (CCK) in bacteria and in insect cells infected with recombinant baculovirus.

Neuropeptide prohormones are generally not abundant in nature as they exits to be processed and contain sites which could be cleaved by a number of cellular proteases. In order to study the processing of prohormones in vitro it is necessary to produce them in quantity. Finding an expression system which produces intact prohormone has been a matter of trial and error. We report that intact rat pro CCK was produced with an amino-terminal His-Tag in e. coli, and it was secreted from sf9 and other insect cells infected with a recombinant baculovirus vector. The bacteria contained about 0.1 micrograms pro CCK/ml of cells. The High 5 insect cells produced 4.3 micrograms/ml medium (as determined by RIA), 10 times as much as sf9 or sf21. Using a combination of ion exchange, gel filtration and HPLC, the insect cell protein was purified about 150 fold with a recovery of about 16%. The secreted insect cell pro CCK is tyrosine sulfated like its mammalian equivalent. Using these expression systems it is possible to produce significant (microgram to mg) quantities of pro CCK for immunologic, enzymatic and structural studies.

Animals↗
PubMed1997

Prohormone convertase 2 is necessary for the formation of cholecystokinin-22, but not cholecystokinin-8, in RIN5F and STC-1 cells.

Two endocrine tumor cell lines from pancreas (RIN5F) and intestine (STC-1) express cholecystokinin (CCK) messenger RNA and are able to posttranslationally process pro-CCK to CCK-22 and CCK-8 amide. Both of these forms are also secreted by these cells. Because they make and secrete forms of amidated CCK larger than CCK-8, they represent a model of pro-CCK processing in the gut and allow investigation of possible mechanisms for tissue differences in prohormone processing. Both of these cells express two endoproteases convertase-1 (PC1) also known as PC3 and prohormone convertase-2 (PC2), which may be involved in pro-CCK processing. We have previously shown than inhibition of PC1 expression in these cells using stable expression of antisense messenger RNA caused a significant reduction in cellular content of amidated CCK and caused a selective depletion of CCK-8 with a comparative sparing of CCK-22. We demonstrate here that inhibition of PC2 expression in these cells also caused a large initial decrease in CCK content and produced a selective depletion of CCK-22 and a comparative sparing of CCK-8. These results support both a role for both PC1 and PC2 in pro-CCK processing in these cells and the hypothesis that tissue-specific processing of pro-CCK may be explained by differences in expression or activity of PC1 and PC2.

Cholecystokinin↗
PubMed1997

Cholecystokinin (CCK) levels are greatly reduced in the brains but not the duodenums of Cpe(fat)/Cpe(fat) mice: a regional difference in the involvement of carboxypeptidase E (Cpe) in pro-CCK processing.

In order to assess the possible role of carboxypeptidase E (Cpe) in pro-CCK processing, tissues from the Cpe(fat)/Cpe(fat) mice were analyzed for CCK content and molecular forms using specific RIAs directed against different portions of the prohormone. Levels of amidated CCK were decreased by about 74% in whole brain of Cpe(fat)/Cpe(fat) mice in comparison to control mice, while levels of amidated CCK in intestine were only reduced by about 36%. In contrast, using an antiserum specific for CCK Gly Arg Arg, Cpe(fat)/Cpe(fat) mice brain had about 13-fold higher levels of this peptide relative to controls, while levels were identical in mutant and control duodenal tissue. This study demonstrates a regional difference in the involvement of Cpe in pro-CCK processing. The accumulation of CCK Gly Arg Arg in Cpe(fat)/Cpe(fat) brains provides definitive proof that the dibasic cleavage of the carboxyl terminus of pro CCK occurs on the carboxyl terminal of the dibasic, between the Arg and Ser as well as confirming that amidated CCK 8 in brain originates from CCK 8 Gly Arg Arg rather than from larger amidated peptides like CCK 22 or CCK 33. The Cpe(fat)/Cpe(fat) mouse phenotype obviously involves multiple endocrine defects, however, it is tempting to speculate that this severe CNS deficiency in CCK 8 may be related to the adult-onset obesity seen in this mutant mouse.

Animals↗
PubMed1996

Cholecystokinin (CCK) release from rat brain slices in vitro is enhanced by agents which elevate intracellular cAMP.

Cholecystokinin (CCK) release from rat brain slices in vitro is enhanced by agents which elevate intracellular cAMP. Treatment of several CCK expressing tumor cells in culture with agents which increase intracellular cAMP increased secretion of CCK. The possibility that elevation of cAMP also alters potassium-evoked release of CCK from rat brain slices incubated in vitro was also investigated. Treatment of slices of rat prefrontal cortex and caudate-putamen with 5 microM forskolin plus 0.5 mM IBMX (3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor) caused a significant elevation of potassium-evoked CCK release. That cAMP can increase CCK release in intact brains slices as well as endocrine tumor cells from thyroid, pancreas, pituitary and intestine suggests that cAMP is a common intracellular mediator for the regulation of CCK release.

1-Methyl-3-isobutylxanthine↗
PubMed1996

Specificity and kinetic studies on the cleavage of various prohormone mono- and paired-basic residue sites by yeast aspartic protease 3.

The specificity and relative efficiency of cleavage of mono- and paired-basic residue processing sites by YAP3p was determined in vitro for a number of prohormone substrates: human ACTH1 39, bovine proinsulin, porcine cholecystokinin 33, cholecystokinin (CCK) 13-33, dynorphin A(1-11), dynorphin B(1-13), and amidorphin. YAP3p generated ACTH1-15 from ACTH1-39. It cleaved proinsulin at the paired-basic residue sites of the B-C junction as well as the C-A junction. Leu-enkephalin-Arg and Leu-enkephalin-Arg-Arg were generated from dynorphin A and dynorphin B, respectively. YAP3p generated Met-enkephalin-Lys-Lys from amidorphin showing that cleavage by this enzyme can occur at a lone pair of Lys residues. CCK33 was cleaved at Lys23 and Arg9, each containing an upstream Arg residue at the P6 and P5 position, respectively. Km values were between 10(-4) and 10(-5) M for the various substrates, with the highest affinity exhibited for the tetrabasic site of ACTH1-39 (1.8 x 10(-5) M). The tetrabasic residue site of ACTH1-39 was cleaved with the highest relative efficiency (kcat/Km = 3.1 x 10(6) m-1 s-1), while that of the monobasic site of CCK13-33 and the paired-basic site of proinsulin B-C junction, were cleaved less efficiently at 4.2 x 10(4) m-1 s-1 and 1.6 x 10(4) m-1 s-1, respectively.

Adrenocorticotropic Hormone↗
PubMed1995

Expression of antisense PC1 in stably transfected RIN5F cells significantly reduces CCK 8 biosynthesis.

The subtilisin-like endoprotease PC1 (PC3) has been implicated in the processing of a number of prohormones. To evaluate whether PC1 may be important for the processing of pro CCK to CCK 8, stable cell lines expressing a portion of the PC1 cDNA in the antisense orientation were established from RIN5F cells. These cells express CCK mRNA, produce and display regulated secretion of CCK 8. One of the clones, R1E8, expresses antisense PC1 mRNA as determined by reverse transcriptase-PCR (RT-PCR) and contains a significantly reduced level of PC1 protein. As compared to both RIN5F and RIN5F control cells (transfected with the expression plasmid containing no antisense message), R1E8 contains only about 30% cell content of CCK 8. These results suggest that PC1 may be important for the processing of CCK 8 from pro CCK.

Animals↗
PubMed1994

Inhibition of pro-cholecystokinin (CCK) sulfation by treatment with sodium chlorate alters its processing and decreases cellular content and secretion of CCK 8.

Pro-cholecystokinin (CCK) has three sulfated tyrosine residues. Sulfation of the tyrosine residue in CCK 8 is known to be important for its activity at CCK A receptors. The role of these sulfated tyrosines in the sorting and processing of pro-CCK was examined by treatment of CCK-secreting rat thyroid medullary carcinoma cells with 10 nM sodium chlorate (a non-toxic inhibitor of tyrosine sulfation). This treatment caused a 50% decrease in the cellular content of immunoreactive CCK and an 80% decrease in its secretion. Sephadex G-50 chromatography of cellular extracts and culture media showed a selective depletion of CCK 8. There was a comparative sparing of CCK 33 and larger molecular forms in cellular extracts which was not observed in the media. These results suggest that the sulfation of the tyrosines of pro-CCK is clearly important for the correct sorting and/or processing of pro-CCK. The pattern of immunoreactive CCK peptides seen with chlorate treatment is consistent with the substrate specificity of a recently identified putative CCK cleaving enzyme and suggests that unsulfated pro-CCK is not efficiently processed to CCK 8 in vivo. The large decrease in CCK content and secretion observed with sodium chlorate may also be due to inefficient sorting of unsulfated pro-CCK into secretory vesicles.

Animals↗