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PubMed1999

Isolation and structural and functional characterization of two stable peptic fragments of human alpha-fetoprotein.

Short-time limited peptic hydrolysis of ligand-free human alpha-fetoprotein (AFP) gave two main fragments with molecular masses of 38 and 32 kDa, which had been produced by splitting of the molecule at the position Leu(312)-Asn(313). A more prolonged proteolysis led to the further degradation of these fragments and appearance of highly proteolytically resistant 23-kDa (P23) and 26-kDa (P26) fragments, corresponding to N- and C-terminal parts of the AFP molecule, respectively. Comparative study of intact free of ligands AFP and isolated stable P23 and P26 fragments by circular dichroism, differential scanning calorimetry, and immunoprecipitation techniques demonstrated that these fragments conserved native secondary, tertiary; and antigenic structure, characteristic of the intact molecule. It was concluded that, free of ligands, the AFP molecule could be considered as a three-domain molecule, in which two compact rigid domains (N-terminal domain I and C-terminal domain III) are connected by relatively labile domain II. The structure of domain II could be approximated by a "molten globule" state, characterized by the absence of rigid tertiary structure but having a pronounced secondary structure. Tumor-suppressive activity via induction of apoptosis was recently shown for AFP [Dudich, E. I., et al. (1998) Tumor Biol. 19, 261-272]. We studied here the ability of isolated proteolytic AFP fragments to induce apoptosis in the AFP-sensitive Raji cell line, to determine possible localization of the active site responsible for apoptosis signaling. Unlike intact AFP, neither isolated fragments nor their equimolar mixture was able to induce apoptosis in a human lymphoma Raji cell line. However, it was demonstrated that both fragments P23 or P26 and their equimolar mixture P23 + P26 operated synergistically with intact AFP in suppression of Raji cell proliferation. These data suggested that two structurally determined requirements are necessary for AFP-mediated triggering of apoptosis: (i) dimerization of AFP to form the heterodimeric complex of C- and N-terminal domains and (ii) participation of the central part of AFP molecule (domain II).

Antigens↗
PubMed2005

Topically applied antibody fragments penetrate into the back of the rabbit eye.

AIMS: Antibody fragments have been shown to penetrate into the anterior chamber when applied to the cornea. The aim of this study was to investigate whether such fragments could penetrate into the vitreous cavity after topical administration to the ocular surface of rabbits. METHODS: An engineered single-chain variable-domain antibody fragment with specificity for an irrelevant rat determinant was applied as a 50 microl eye drop to the eyes of live rabbits at 20-min intervals over 12 h. Eye drops contained 0.8-1.1 mg/ml protein in a buffered salt solution supplemented with penetration and viscosity enhancers. Samples were collected by paracentesis from the vitreous cavity immediately postmortem. Antibody fragments in these samples were quantified by measuring the binding activity to specific antigen, using flow cytometry. RESULTS: Topically applied antibody fragments were detectable in the vitreous of rabbit eyes after 4-12 h but had cleared at 12 h following the final eye drop. Concentrations of the antibody fragment in the vitreous samples were estimated to be 50-150 ng/ml at 12 h. Penetration of the parental whole antibody into the vitreous was not observed. CONCLUSION: Antibody fragments penetrate into the vitreous chamber of the rabbit eye after topical administration to the ocular surface. Such fragments may have therapeutic potential for diseases affecting the posterior segment.

Administration, Topical↗
PubMed1974

Purification and characterization of the seven cyanogen bromide fragments of human serum transferrin.

1. Procedures are described for the isolation of seven distinct cyanogen bromide fragments in high yield from human serum transferrin. 2. Cyanogen bromide-cleaved transferrin is separated into three fragments (CN-A, CN-B and CN-C) by gel filtration with Sephadex G-100. 3. Four peptides are obtained from CN-A (the largest fragment) after reduction and carboxamidomethylation, by gel filtration in acidic solvents. Two peptides are similarly obtained from fragment CN-B, whereas fragment CN-C is a single cystine-free peptide. 4. The molecular weights of the seven peptides, as determined by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate, by sedimentation-equilibrium ultracentrifugation and by sequence studies, range from 3100 to 27000. Together they account for a molecular weight of 76200 for transferrin. 5. The two largest fragments contain the carbohydrate attachment sites of the protein, and the smallest fragment is derived from the N-terminus. 6. The amino acid compositions and N-terminal groups of the fragments are reported and the results compared with those of previous investigations.

Amino Acid Sequence↗
PubMed1979

The effects of calcium ions and pH on bovine prothrombin fragment 1. Intrinsic fluroescence studies.

The effects of pH and Ca2+ on the intrinsic fluorescence of bovine prothrombin fragment 1 were investigated to deduce the nature of protein functional groups involved in Ca2+ binding to fragment 1. From pH values of 9 to 3, increasing the H3O+ concentration results in quenching of the fluorescence of fragment 1. Reversible pH-titration curves are obtained which appear to consist of two regions. From pH 4 to pH6.5 a broad titration curve is obtained, whereas from pH6.5 to 9 a more pronounced titration behaviour is evidenced by a group or groups on fragment 1 with an apparent pKa of approx. 7.5. In contrast, the apparent association constant for Ca2+ and fragment 1 shows a sharp pH-dependence in the region between pH7 and 8 with tighter Ca2+ binding at higher pH values. A PKa of approx. 7.5 can be estimated for the group or groups on fragment 1 linked to the tight binding of Ca2+. Both H3O+ and Ca2+ result in blue-shifts in the wave-lengths of fragment-1 emission. These results are interpreted in terms of H+ - and Ca2+ - induced changes in the conformation of fragment 1 as a result of surface-charge neutralization.

Animals↗
PubMed1979

The anion-transport protein of the human erythrocyte membrane. Studies on fragments produced by pepsin digestion.

We have studied the fragmentation by pepsin in 1 M-acetic acid of the erythrocyte anion-transport protein in erythrocyte membranes. The location of the fragments obtained was determined by radioiodinating the protein with the use of lactoperoxidase, and identifying the labelled peptides obtained in peptide "maps" of thermolysin digests of the fragments. Three of the fragments were found to be related overlapping products, and shared a common C-terminus. The major site of pepsin cleavage leading to the C-termini of these fragments was shown to be close to the major site of extracellular cleavage of the protein by proteinases active at a neutral pH. Another two fragments were isolated and shown to be derived from the C-terminal portion of the protein. No well-defined large radioactive fragments of the protein were solubilized from the membrane by pepsin in 1 M-acetic acid, the bulk of the radioactivity attributable to the anion transport protein being recovered in very small fragments that could not be resolved by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Our results suggest that the polypeptide chain of the anion-transport protein emerges at the extracellular face of the membrane 8000-13000 daltons on the N-terminal side of the major site of extracellular cleavage of the protein by proteinases that are active at a neutral pH.

Amino Acid Sequence↗
PubMed1984

Immunologically reactive tryptic fragments of human prostatic acid phosphatase.

Three peptide fragments (designated II, III and IV) of human prostatic acid phosphatase (PAP) were isolated to homogeneity from a limited tryptic hydrolysate of PAP by gel filtration on Sephadex G-100, followed by chromatography on DEAE-cellulose and Sephadex G-75. The homogeneity was confirmed by disc poly-acrylamide-gel electrophoresis. The Mr values were 32 500, 25 000 and 11 000 as estimated by gel filtration and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Immunoprecipitation study revealed that only fragment II formed an immune precipitate with anti-PAP antibodies. Fragment II exhibited 45% of maximum inhibitory activity on the reaction between PAP and goat anti-PAP IgG (immunoglobulin G) antibodies (or rabbit anti-PAP antibodies), whereas fragments III and IV demonstrated 24% (or 23%) and 29% (or 27%) inhibition respectively. A mixture of these three tryptic fragments of PAP result in 96% (for goat anti-PAP antibodies) and 94% (for rabbit anti-PAP antibodies) inhibitory activities, which were equivalent to the sum of maximum inhibitory activity of the three fragments individually. The results demonstrated that these three tryptic peptide fragments carried all the antigenic active sites of the native PAP, and suggested that the entire molecule of human PAP comprised a minimum of four distinguishable, nonoverlapping antigenic determinants. These three fragments also were shown to retain all the disulphide bonds of the native PAP, and thus were useful reagents for the elucidation of PAP molecular structure.

Acid Phosphatase↗
PubMed1992

Association between diphtheria toxin A- and B-fragment and their fusion proteins.

Natural diphtheria toxin is synthesized as a single polypeptide chain that is activated by cleavage into an A- and a B-fragment, which are linked by a disulphide bond. In the present work the ability of independently translated A- and B-fragments to associate was investigated. Low amounts of A- and B-fragments synthesized in vitro were mixed under conditions that allowed formation of a disulphide bridge between the fragments. Under these conditions toxin was reconstituted in close to 100% yield and found to be as toxic to Vero cells as natural diphtheria toxin. Efficient association between the A- and B-fragment was dependent on the formation of a disulphide bridge. Reconstituted toxin obtained from one [35S]methionine-labelled fragment and one unlabelled fragment proved useful in translocation studies. Addition of a number of different polypeptides to the N- and C-termini of either fragment did not, in most cases, prevent reconstitution. The ready reconstitution allows easy manipulations with the toxin to form targeted molecules and to develop diphtheria toxin as a vector for translocation of peptides to the cytosol. The fact that the reconstituted toxin does not need to be nicked with proteinases to be active allows experimentation with proteinase-sensitive constructs.

Amino Acid Sequence↗
PubMed1998

Complementation studies with co-expressed fragments of human red cell band 3 (AE1): the assembly of the anion-transport domain in xenopus oocytes and a cell-free translation system.

We examined the assembly of the membrane domain of the human red cell anion transporter (band 3; AE1) by co-expression of recombinant N- and C-terminal fragments in Xenopus oocytes and in cell-free translation with canine pancreatic microsomes. Co-immunoprecipitation was performed in non-denaturing detergent solutions using antibodies directed against the N- and C-termini of the membrane domain. Eleven of the twelve fragments were expressed stably in oocytes in the presence or absence of their respective partners. However, the fragment containing from putative span nine to the C-terminus could be detected in oocytes only when co-expressed with its complementary partner containing the first eight spans. Co-expression of pairs of fragments divided in the first, second, third and fourth exofacial loops and in the fourth cytoplasmic loop resulted in a concentration-dependent association, but a pair of fragments divided in the sixth cytoplasmic loop did not co-immunoprecipitate. When two complementary fragments were translated separately in the cell-free system and the purified microsomes were then mixed, co-immunoprecipitation was observed only if the membranes were first fused using polyethylene glycol. This shows that co-immunoprecipitation results from specific interactions within the membrane and is not an artefact of detergent solubilization or immunoprecipitation. We demonstrate that band 3 assembly can occur within the membrane after translation, insertion and initial folding of the individual fragments have been completed. We conclude that most band 3 fragments contain the necessary information to fold in the membrane and adopt a structure that is sufficiently similar to the native protein that it permits correct assembly with its complementary partner.

Animals↗
PubMed1995

Calcitonin gene-related peptide as an endogenous vasodilator: immunoblockade studies in vivo with an anti-calcitonin gene-related peptide monoclonal antibody and its Fab' fragment.

1. Calcitonin gene-related peptide (CGRP) is localized in perivascular sensory neurons and is a potent vasodilator. We investigated the utility of immunoblockade as an in vivo technique for probing the role of CGRP as an endogenous vasodilator. 2. The effects of an anti-CGRP monoclonal antibody (MAb; coded C4.19) and its Fab' fragment on CGRP-induced changes in blood pressure and skin blood flow were studied in pentobarbitone-anaesthetized rats. Antidromic skin vasodilatation in the rat hind paw was measured by laser Doppler fluxmetry. 3. The dose-response relationship for the hypotensive effect of intravenous rat alpha CGRP (r alpha CGRP) was similarly shifted rightward by MAb C4.19 IgG (1 mg/rat; intravenously) and Fab' fragment (2 mg/rat; intravenously). The C-terminal fragment of human alpha CGRP (h alpha CGRP8-37) also blocked the hypotensive effect of r alpha CGRP. 4. MAb C4.19 Fab' fragment (2 mg/rat; intravenously) and h alpha CGRP8-37 (100 nmol/kg; intravenously), but not MAb C4.19 IgG (up to 3 mg/rat; intravenously) or normal mouse Fab' fragment (2 mg/rat; intravenously), blocked the increased skin blood flow response to antidromic stimulation of the saphenous nerve. 5. The mean percentage changes in skin blood flow parameters due to MAb C4.19 Fab' fragment were significantly different from those due to normal mouse Fab' fragment (unpaired t-test; P < 0.05) but not from those due to h alpha CGRP8-37. 6. The results demonstrate the pharmacokinetic advantage of Fab' fragment over IgG for immunoblockade studies in vivo and support the role of CGRP in mediating skin vasodilatation.

Animals↗
PubMed1998

The C-terminal fragment of big endothelin-1 does not potentiate the vasoactive effects of endothelin-1.

The aim was to study the cardiovascular effects of the C-terminal (22-38) fragment of big endothelin-1, which is produced by the cleavage of big endothelin-1 (big ET-1) to endothelin-1 (ET-1). An intravenous infusion of the (22-38) fragment (4, 8 and 12 pmol kg-1 min-1, each dose for 10 min) was given to 10 healthy subjects. Four control subjects received 0.9% saline. Two additional subjects received ET-1 1 (0.2 and 4 pmol kg-1 min-1, each dose for 20 min) alone or combined with an equimolar infusion of the (22-38) fragment on two separate occasions. The fragment infusion did not alter heart rate, mean arterial blood pressure, cardiac output, systemic or pulmonary vascular resistance, splanchnic, cerebral or forearm blood flow. Renal blood flow showed a slight fall (11%, P < 0.001) in the fragment group of the same magnitude as in a previous control study. After the fragment infusion, a decrease in mean pulmonary arterial pressure (MPAP) by 12% (P < 0.01) and in pulmonary capillary wedge pressure (PCWP) by 31% (P < 0.001) was noted, which did not differ from the pulmonary pressures in the saline-infused control group. The (22-38) fragment, when combined with ET-1, was not able to modify the effects of ET-1 on heart rate, mean arterial blood pressure, splanchnic and renal blood flow. Consequently, the exogenous (22-38) fragment does not seem to cause any significant cardiovascular effects in healthy humans.

Adult↗
PubMed2000

Isolation and characterization of recombinant antibody fragments against CDC2a from Arabidopsis thaliana.

In order to obtain recombinant antibody fragments that bind the cell-cycle protein CDC2a from Arabidopsis thaliana (CDC2aAt), two phage display libraries of single-chain variable (scFv) fragments were constructed. One library was derived from mice immunized with recombinant CDC2aAt N-terminally fused to a His6-tag (His-CDC2aAt) and the other was made out of an anti-PSTAIRE hybridoma cell line. Six specific His-CDC2aAt-binding phage clones (3D1, 3D2, 3D10, 3D25, 4D21 and 4D47) were isolated by panning. The isolated monoclonal phage clones, as well as the soluble scFv fragments produced in the periplasm of Escherichia coli, bind His-CDC2aAt in ELISA and on Western blots. Moreover, four clones (3D1, 3D2, 3D10 and 4D21) detect specifically CDC2aAt from Arabidopsis cell suspensions on Western blots. Clone 4D21 binds the PSTAIRE epitope, whereas the 3D1, 3D2 and 3D10 clones bind, as yet unidentified, epitopes of CDC2aAt. Furthermore, the accumulation and antigen-binding activity of these scFv fragments in a reducing environment were assessed. No interaction could be shown between the scFv fragments and CDC2aAt in a yeast two-hybrid assay. However, after transient expression of the scFv fragments in the cytosol of tobacco leaves, three of six scFv fragments (3D1, 3D2 and 3D10) accumulated in the plant cytosol and ELISA results indicate that these scFv fragments retained antigen-binding activity.

Amino Acid Sequence↗
PubMed1999

Controlled cleavage of KLH1 and KLH2 by the V8 protease from Staphylococcus aureus reassociation, electrophoretic and transmission electron microscopy study of peptide fragments.

The reassociation behaviour of protease V8-cleaved peptides from KLH1 and KLH2, the two hemocyanin isoforms from the giant keyhole limpet Megathura crenulata, has been studied by transmission electron microscopy of negatively stained specimens and SDS/PAGE. Reassociation of the complete mixture of protease cleavage products and of combinations of peptide fragments purified by HPLC was performed in the presence of 100 mm CaCl2 and 100 mm MgCl2 at pH 7.4, over a period of 1 to 4 weeks. The V8 protease splits KLH1 into peptide fragments containing the functional units abc, def, defg, defgh, g and h. This mixture of peptide fragments reassociated to form helical tubular polymers, with a diameter of approximately 25 nm. The single functional units g and h were not incorporated into the polymer. An essentially identical polymer was formed from the re-mixed HPLC-purified fragments abc, def and defg alone. As with uncleaved subunit, the tubular polymer of V8-cleaved KLH1 forms bundles. The combination of peptides def and defg led to the formation of short arc-like filamentous structures, which aggregated but showed little tendency to associate into larger polymers. The KLH1 peptide fragments abc and def alone, did not reassociate and in combination their potential to form polymers was very low. With KLH2, the V8 protease generated peptide fragments containing the functional units abc, defg, defgh and h, which in combination slowly reassociated to form a tubular polymer significantly different to that obtained from the KLH1 V8 fragments. The three-functional unit fragment abc from KLH2 showed no tendency to polymerize and the combination of peptides defg + defgh generated only disordered aggregates, with some indication of malformed tubules. The combination of biochemical and electron microscopical methods enabled the characterization of these polymers with respect to peptide composition and higher order structure.

Electrophoresis, Polyacrylamide Gel↗
PubMed2001

Heat capacity analysis of oxidized Escherichia coli thioredoxin fragments (1--73, 74--108) and their noncovalent complex. Evidence for the burial of apolar surface in protein unfolded states.

We have calculated the absolute heat capacities of fragments 1--73 (N fragment) and 74--108 (C fragment) from thioredoxin, their complex and the uncleaved protein, from the concentration dependence of the apparent heat capacities of the solutions determined by differential scanning calorimetry. We find that, while the absolute heat capacities of uncleaved, unfolded thioredoxin and the C fragment are in good agreement with the theoretical values expected for fully solvated chains (calculated as the sum of the contributions of the constituent amino acids), the absolute heat capacities of the N fragment and the unfolded complex are about 2 kJ x K(-1) x mol(-1) lower than the fully solvated-chain values. We attribute this discrepancy to burial of the apolar surface in the N fragment (as burial of the polar area is expected to lead to an increase in heat capacity). Illustrative calculations suggest that burial of about 1000--1600 A(2) of apolar surface takes place in the N fragment (probably accompanied by the burial of a smaller amount of polar surface). In general, this work is supportive of heat capacity measurements on protein fragments being useful as probes of surface burial in studies to characterize protein unfolded states and the high regions of protein folding landscapes.

Buffers↗
PubMed2001

Generation of a constitutively active fragment of PKN in microglia/macrophages after middle cerebral artery occlusion in rats.

PKN is a fatty acid- and Rho-activated serine/threonine kinase, which has a catalytic domain highly homologous to that of protein kinase C (PKC). Recent studies have demonstrated that PKN is proteolytically cleaved after apoptotic stimulation and then a constitutively active 55-kDa fragment is generated. However, the role of the 55-kDa fragment are poorly understood. Adult Sprague-Dawley (SD) rats underwent middle cerebral artery occlusion (MCAO), and the temporal and spatial changes in the fragmentation of PKN and of PKC delta were examined by immunoblotting. No proteolytic fragment of PKC delta (about 40 kDa) was detected. The 55-kDa fragment of PKN appeared transiently from 3 days after MCAO at the ipsilateral normal cortex. At the boundary zone of infarction, the 55-kDa fragment was markedly induced from day 5 then peaked on day 21 and persisted until day 28. Analysis of anti-phosphoserine immunoprecipitates with an anti-PKN antibody revealed phosphorylation of the 55-kDa band. Double staining for PKN and Ox42 was used to examine the source of the 55-kDa fragment. PKN immunoreactivity was significantly increased in Ox42-positive cells (microglia/hematogenous macrophages). No DNA laddering and only a few terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive cells were observed on day 14 in despite of the high level appearance of the 55-kDa band. These results suggest that the constitutively active 55-kDa fragment of PKN does not contribute to apoptosis, but may contribute to a function of microglia/macrophages.

Animals↗
PubMed1984

Binding of alpha-bungarotoxin to proteolytic fragments of the alpha subunit of Torpedo acetylcholine receptor analyzed by protein transfer on positively charged membrane filters.

Proteolytic fragments of the alpha subunit of the acetylcholine receptor retain the ability to bind alpha-bungarotoxin following resolution by polyacrylamide gel electrophoresis and immobilization on protein transfers. The alpha subunit of the acetylcholine receptor of Torpedo electric organ was digested with four proteases: Staphylococcus aureus V-8 protease, papain, bromelain, and proteinase K. The proteolytic fragments resolved on 15% polyacrylamide gels were electrophoretically transferred onto positively charged nylon membrane filters. When incubated with 0.3 nM 125I-labeled alpha-bungarotoxin and autoradiographed, the transfers yielded patterns of labeled bands characteristic for each protease. The molecular masses of the fragments binding toxin ranged from 7 to 34 kDa, with major groupings in the 8-, 18-, and 28-kDa ranges. The apparent affinity of the fragments for alpha-bungarotoxin as determined from the IC50 value was 6.7 X 10(-8) M. The labeling of fragments with alpha-bungarotoxin could be inhibited by prior affinity alkylation of receptor-containing membranes with 4-(N-maleimido)-alpha-benzyltrimethylammonium iodide. These findings demonstrate that immobilized proteolytic fragments as small as 1/5 the size of the alpha subunit retain the structural characteristics necessary for binding alpha-bungarotoxin, although the toxin is bound to the fragments with lower affinity than to the native receptor. The effect of affinity ligand alkylation demonstrates that the alpha-bungarotoxin binding site detected on the proteolytic fragments is the same as the affinity-labeled acetylcholine binding site on the intact acetylcholine receptor.

Animals↗
PubMed1988

95- and 25-kDa fragments of the human immunodeficiency virus envelope glycoprotein gp120 bind to the CD4 receptor.

125I-labeled gp120 (120-kDa envelope glycoprotein) from the BH10 isolate of human immunodeficiency virus is cleaved to a limited extent with the glutamate-specific protease from Staphylococcus aureus. After disulfide bond reduction, fragments with approximate molecular masses of 95, 60, 50, and 25 kDa are produced. Tests for binding to CD4-positive cells show that only two fragments, the 95- and 25-kDa peptides, are observed in cleavage products that retain the selective binding capacity of gp120. Radiosequence analysis of the fragments after sodium dodecyl sulfate/polyacrylamide gel electrophoresis and electroblotting demonstrates that the 95-kDa fragment lacks the N-terminal region of gp120 and starts at position 143 of the mature envelope protein. The 50-kDa fragment starts at the same position. The 25-kDa binding fragment was similarly deduced to be generated as a small fragment from a cleavage site in the C-terminal part of gp120. The identifications of these fragments demonstrate that radiosequence analysis utilizing 125I-labeled tyrosine residues can function as a useful and reliable method for small-scale determination of cleavage sites in proteins. Combined, the data suggest domain-like subdivisions of gp120, define at least two intervening segments especially sensitive to proteolytic cleavage, and demonstrate the presence of a functional region for receptor binding in the C-terminal part of the molecule.

Cell Line↗
PubMed2001

A novel gamma -secretase assay based on detection of the putative C-terminal fragment-gamma of amyloid beta protein precursor.

Alzheimer's disease is characterized by the deposits of the 4-kDa amyloid beta peptide (A beta). The A beta protein precursor (APP) is cleaved by beta-secretase to generate a C-terminal fragment, CTF beta, which in turn is cleaved by gamma-secretase to generate A beta. Alternative cleavage of the APP by alpha-secretase at A beta 16/17 generates the C-terminal fragment, CTFalpha. In addition to A beta, endoproteolytic cleavage of CTF alpha and CTF beta by gamma-secretase should yield a C-terminal fragment of 57-59 residues (CTF gamma). However, CTF gamma has not yet been reported in either brain or cell lysates, presumably due to its instability in vivo. We detected the in vitro generation of A beta as well as an approximately 6-kDa fragment from guinea pig brain membranes. We have provided biochemical and pharmacological evidence that this 6-kDa fragment is the elusive CTF gamma, and we describe an in vitro assay for gamma-secretase activity. The fragment migrates with a synthetic peptide corresponding to the 57-residue CTF gamma fragment. Three compounds previously identified as gamma-secretase inhibitors, pepstatin-A, MG132, and a substrate-based difluoroketone (t-butoxycarbonyl-Val-Ile-(S)-4-amino-3-oxo-2, 2-difluoropentanoyl-Val-Ile-OMe), reduced the yield of CTF gamma, providing additional evidence that the fragment arises from gamma-secretase cleavage. Consistent with reports that presenilins are the elusive gamma-secretases, subcellular fractionation studies showed that presenilin-1, CTF alpha, and CTF beta are enriched in the CTF gamma-generating fractions. The in vitro gamma-secretase assay described here will be useful for the detailed characterization of the enzyme and to screen for gamma-secretase inhibitors.

Alzheimer Disease↗
PubMed2000

Functional competition between poly(ADP-ribose) polymerase and its 24-kDa apoptotic fragment in DNA repair and transcription.

Poly(ADP-ribose) polymerase is a 113-kDa nuclear enzyme that binds to both damaged DNA and to RNA associated with actively transcribed regions of chromatin. Binding of poly(ADP-ribose) polymerase to DNA lesions activates it, catalyzing the covalent addition of multiple ADP-ribose polymers to the enzyme (automodification). During apoptosis, poly(ADP-ribose) polymerase is cleaved by caspase-3, resulting in the formation of an N-terminal 24-kDa fragment, containing the DNA binding domain, and a C-terminal 89-kDa catalytic fragment. The functional relevance of this cleavage is not well understood. We therefore prepared a recombinant 24-kDa poly(ADP-ribose) polymerase fragment and investigated the role of this fragment in DNA repair and transcription. The 24-kDa fragment retained its binding affinity for both DNA breaks and RNA. In an in vitro cell-free DNA repair assay, this fragment inhibited rejoining of DNA breaks and suppressed ADP-ribose polymer formation by competing with poly(ADP-ribose) polymerase in binding to DNA breaks. With regard to transcription, it has recently been demonstrated that binding of poly(ADP-ribose) polymerase to transcribed RNA reduces the rate of transcript elongation and that automodification of poly(ADP-ribose) polymerase bound to DNA breaks results in up-regulation of transcription. We tested the 24-kDa fragment for its ability to suppress transcript elongation, and we found that it competed against the up-regulation of transcription mediated by full-length poly(ADP-ribose) polymerase. The ability of the 24-kDa fragment to inhibit DNA repair, ADP-ribose polymer formation, and damage-dependent up-regulation of transcription may contribute to the apoptotic shift from cell survival to cell death mode.

Apoptosis↗