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M C Peitsch

Publications and source records attributed to M C Peitsch.

At least 55 records · Page 3Linked to original sources
PubMed1995

Characterization of the non-functional Fas ligand of gld mice.

Mice homozygous for either the gld or lpr mutation develop autoimmune diseases and progressive lymphadenopathy. The lpr mutation is characterized by the absence of functional Fas, whereas gld mice exhibit an inactive FasL due to a point mutation proximal to the extracellular C-terminus. The structural repercussions of this amino acid substitution remain unknown. Here we report that FasL is expressed at similar levels on the surface of activated T lymphocytes from gld and wild-type mice. Using a polyclonal anti-FasL antibody, indistinguishable amounts of a 40 kDa protein are detected in both gld and wild-type splenocytes. The molecular model of FasL, based on the known structure of TNF-alpha, predicts that the Phe --> Leu gld mutation is located at the protomer interface which is close to the FasR interaction site. We conclude that the gld mutation allows normal FasL biosynthesis, surface expression and oligomerization, but induces structural alterations to the Fas binding region leading to the phenotypic changes observed.

Amino Acid Sequence↗
PubMed1994

Mutational analysis of polymeric immunoglobulin receptor/ligand interactions. Evidence for the involvement of multiple complementarity determining region (CDR)-like loops in receptor domain I.

The polymeric Ig receptor (pIgR) mediates the transport of IgA and IgM across a variety of mucosal epithelia. The ectodomain of this receptor consists of five immunoglobulin-like domains (I-V), the first four being structurally similar to immunoglobulin variable regions, and the fifth to Ig constant regions. This study examines the structural features of the pIgR that participate in binding of the ligand, dimeric IgA (dIgA). Recent evidence suggests that a highly conserved region of the first Ig-like domain (domain I) may be important in this process (Bakos, M.A., Kurosky, A., and Goldblum, R. M. (1991) J. Immunol. 147, 3419-3426). In support of this hypothesis, molecular modeling of domain I places this conserved region in an exposed loop analogous to the CDR1 loop of Ig, suggesting that interactions between dIgA and the pIgR may be similar to those between antibodies and their cognate antigens. To test this hypothesis directly, we performed a mutagenic analysis of all three CDR-like loops in domain I of the pIgR. We found that point mutations in multiple residues of CDR1 produced effects on IgA binding ranging from minimal (90% of control) to profound (7%). In addition, we replaced regions corresponding to the CDR2 and CDR3 loops of domain I with their counterparts from domain II (which does not bind IgA), which in both cases resulted in complete abrogation of IgA binding. Taken together, these data suggest that each of the three CDR-like loops of domain I of the rabbit pIgR participates in the binding of dimeric IgA.

Amino Acid Sequence↗
PubMed1994

Acidic residues in extracellular loops of the human Y1 neuropeptide Y receptor are essential for ligand binding.

To investigate whether negatively charged residues of the human Y1 neuropeptide Y (NPY) receptor are required for ligand binding, a series of mutants were constructed in which aspartic acid and glutamic acid residues present in putative extracellular domains of the Y1 receptor were systematically replaced by alanines. The mutant cDNAs were transiently expressed in HeLa cells using a vaccinia virus-derived expression system, and their ability to bind NPY was evaluated. The level of expression of mutants unable to bind NPY was also tested immunologically. In addition, the ability of the mutant proteins to be recruited to the cell surface was assessed by confocal microscopy. Substitution of aspartic acids and glutamic acids of the N-terminal first extracellular domain had no effect on binding. On the other hand, substitution of acidic residues present in the second, third, and fourth extracellular loops resulted in proteins unable to bind 125I-NPY. These results demonstrate that the extracellular loops of the human Y1 NPY receptor are essential portions of its ligand binding domain.

Amino Acid Sequence↗
PubMed1994

Granzyme B.

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Amino Acid Sequence↗
PubMed1994

The apoptosis endonucleases: cleaning up after cell death?

The term apoptosis describes the predictable structural changes associated with many forms of programmed cell death. One of the first visible events of apoptosis is the collapse of the nucleus. Nuclear degradation is manifested by digestion of chromatin into nucleosome-sized fragments or multiples of these. This digestion of DNA is enzymatic, and several attempts have been made to characterize apoptosis-specific endodeoxyribonucleases. Although there are strong candidates for such enzymes, direct evidence for their role in apoptosis is yet to be provided.

Journal Article↗
PubMed1994

About the involvement of deoxyribonuclease I in apoptosis.

Cell death by apoptosis is involved in a large variety of developmental events and physiological processes requiring a reduction in cell count. Nuclear collapse, one of the first visible changes denoting irreversible commitment to cell death by apoptosis, is frequently accompanied by chromatin degradation into nucleosome-sized fragments of multiples thereof. The identity of the endonuclease responsible for this DNA digestion has attracted some interest in recent years and several candidate endonucleases have been proposed. The scope of this article is to summarise the present knowledge about deoxyribonuclease I, one of the candidate enzymes.

Journal Article↗
PubMed1994

Distribution of deoxyribonuclease I in rat tissues and its correlation to cellular turnover and apoptosis (programmed cell death).

The expression of deoxyribonuclease I (DNase I) in various rat tissues was screened by use of a cDNA-probe of rat parotid DNase I and monospecific polyclonal antibodies. High amounts of DNase I-specific mRNA were found in the parotid gland, kidney and small intestine. Homogenates of these organs also contain elevated levels of DNase I-specific DNA-degrading activity as verified by the zymogram technique and immunoblots. Affinity-purified polyclonal antibodies against rat parotid DNase I were employed in an immunohistochemical study of the cellular distribution of DNase I antigen in rat parotid gland, kidney, small intestine, and a number of stratified epithelia. In the parotid gland the DNase I antigenicity was found to be confined to the secretory cells. Within these cells the secretory granules exhibit the highest immunoreactivity. In contrast, within the small intestine and stratified epithelia we found a preferential localization and concentration of DNase I in cells prone to undergo apoptosis (programmed cell death), i.e., within the migrating enterocytes present at the villar tips and the keratinocytes above the basal cell layer. Within the kidney, the cells lining the convoluted distal tubules and collecting ducts exhibit strong DNase I immunoreactivity which was found to often localize perinuclearly. The cells exhibiting chromatin fragmentation were identified on paraffin-embedded sections by in situ end-labeling of free 3'-OH-ends of cleaved DNA using fluorescent dATP or dUTP and terminal transferase. It was found that only a small fraction of the DNase I positive cells showed signs of apoptotic chromatin degradation. Thus only a few enterocytes at the uppermost villar tips and very few keratinocytes underneath the keratinized layer were in situ end-labeled, i.e., exhibited a high concentration of fragmented DNA. This result is taken as evidence that these cells express DNase I in advance of their apoptotic death and furthermore that the actual apoptosis is a rapid process only detectable in a few cells. In contrast, no in situ end-labeled apoptotic nuclei were detected in rat kidney provided that care was taken to rapidly excise and fix this organ.

Amino Acid Sequence↗
PubMed1993

Hydrophobic C-terminal amino acids in the beta-subunit are involved in assembly with the alpha-subunit of Na,K-ATPase.

To define the structural basis of oligomerization for the alpha- and beta-subunits of Na,K-ATPase, we have attempted to identify the amino acids in the C-terminus of the beta-subunit that are involved in subunit assembly. We predicted that the last 10 amino acids form a beta-strand-like structure exposing on one side a hydrophilic and on the other side a continuous hydrophobic domain. The relative importance of the two domains in assembly was probed by introducing point mutations in either domain of Xenopus beta 3-subunits and by testing the ability of these mutants to stabilize newly synthesized alpha-subunits expressed in Xenopus oocytes and to form functional alpha-beta complexes at the plasma membrane. All single and double mutants with changes at R268 and/or K272 to either uncharged or negatively charged amino acids associated with coexpressed alpha-subunits and increased the number of ouabain binding sites and Rb uptake into oocytes. On the other hand, mutations affecting the hydrophobic amino acids influenced the assembly efficiency with alpha-subunits to a variable extent. The single mutants V269N and I275N did not influence and the mutant V273N slightly affected the assembly process. On the other hand, the cellular accumulation of alpha-subunits and the expression of functional Na,K pumps was considerably reduced with the mutant F271N and totally abolished with the double mutant V269N/F271N. Finally, replacement of V269 and F271 or V273 and I275 with the less hydrophobic alanine also significantly decreased subunit assembly, which was no longer detectable after replacement of all four amino acids.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

DNA fragmentation during apoptosis is caused by frequent single-strand cuts.

One of the hallmarks of apoptosis is the digestion of genomic DNA by an endonuclease, generating a ladder of small fragments of double-stranded DNA. We have examined the nature of the DNA breaks produced in mouse thymocytes triggered to undergo apoptosis by steroids or by stimulation of the T cell receptor. Whereas the typical ladder pattern of oligonucleosomal fragments was observed after agarose gel electrophoresis, numerous single-strand cuts were detected after electrophoresis under denaturing conditions. Single-strand nicks were found to be very frequent in the internucleosomal regions, but also to occur in the core particle-associated DNA. An identical pattern of single-strand nicks was obtained when chromatin DNA was exposed to the single-strand cleaving deoxyribonuclease I. The nicked DNA fragments, extracted from apoptotic thymocytes, were sensitive to the action of S1-nuclease. We propose that DNA fragmentation induced during apoptosis is not due to a double-strand cutting enzyme as previously postulated, but rather is the result of single-strand breaks. This ensures the dissociation of the DNA molecule at sites where cuts are found within close proximity.

Animals↗
PubMed1993

Characterization of the endogenous deoxyribonuclease involved in nuclear DNA degradation during apoptosis (programmed cell death).

Cell death by apoptosis occurs in a wide range of physiological events including repertoire selection of lymphocytes and during immune responses in vivo. A hallmark of apoptosis is the internucleosomal DNA degradation for which a Ca2+,Mg(2+)-dependent endonuclease has been postulated. This nuclease activity was extracted from both rat thymocyte and lymph node cell nuclei. When incubated with nuclei harbouring only limited amounts of endogenous nuclease activity, the ladder pattern of DNA fragments characteristic of apoptosis was induced. This extractable nucleolytic activity was immunoprecipitated with antibodies specific for rat deoxyribonuclease I (DNase I) and was inhibited by actin in complex with gelsolin segment 1, strongly pointing to the presence of a DNase I-type enzyme in the nuclear extracts. COS cells transiently transfected with the cDNA of rat parotid DNase I expressed the enzyme, and their nuclei were able to degrade their DNA into oligosome-sized fragments. PCR analysis of mRNA isolated from thymus, lymph node cells and kidney yielded a product identical in size to that from rat parotid DNase I. Immunohistochemical staining with antibodies to rat DNase I confirmed the presence of DNase I antigen in thymocytes and lymph node cells. The tissue distribution of DNase I is thus extended to tissues with no digestive function and to cells which are known to be susceptible to apoptosis. We propose that during apoptosis, an endonuclease indistinguishable from DNase I gains access to the nucleus due to the breakdown of the ER and the nuclear membrane.

Animals↗
PubMed1993

Mutations in the putative lipid-interaction domain of complement C9 result in defective secretion of the functional protein.

Complement protein C9 assembles with C5, C6, C7, C8 on the surface of target cells to form the lytic membrane attack complex (MAC). During MAC assembly and insertion into the target membrane, the hydrophilic, globular C9 partially unfolds to expose a hydrophobic lipid interaction domain. Several copies of amphiphilic C9 subsequently polymerize to form the characteristic ring-like MAC. Using a combined photoaffinity label and computer modeling approach, two amphipathic helices in a segment encompassing the amino acids 293-334 have been predicted to interact with membrane lipids. To elucidate the mechanism of C9 lipid binding and insertion, site-directed mutagenesis was used to change the amphipathic character of the helices. While some conservative amino acid replacements such as Thr307 by a Leu were tolerated and yielded fully active C9 when expressed in COS cells, successive changes of Leu305 into Val, Ala, and Glu on the hydrophobic site of the first helix gave rise to only partly or not secreted C9. All non-conservative amino acid replacements introduced on either side of the helices resulted in non-secreted C9 that was subsequently degraded intracellularly, indicating the importance of the correct folding of the presumptive transmembrane domain during biosynthesis. A natural secretion-incompetent mutant was found in which Val293, located in the proposed lipid-binding region, was lacking. Taken together, these findings suggest that the high incidence of homozygous C9 deficiencies may be due to a blockage in intracellular transport and secretion due to point mutations in this 'hot spot' region of the molecule.

Affinity Labels↗
PubMed1993

A 3-D model for the CD40 ligand predicts that it is a compact trimer similar to the tumor necrosis factors.

Based on the similarity in primary structure between the newly characterized ligand for CD40 (CD40L) and the tumor necrosis factors (TNFs), we have modeled a detailed 3-D structure for CD40L. We used the known structure of TNF alpha as a template for the generation of the CD40L model. The soundness of the model-building algorithms was verified by constructing a 3-D model of TNF beta and comparing it to its crystallographically determined structure. The CD40L sequence is entirely compatible with the 'jelly-roll' beta-strand structure characteristic of the TNFs. Like the TNFs, CD40L is predicted to form a compact trimer, although the interactions between monomers are distinct from those found in the TNFs. The model predicts which regions of CD40L could interact with its receptor(s) and which amino acids are essential for the maintenance of its trimeric structure.

Amino Acid Sequence↗
PubMed1993

Overexpression of deoxyribonuclease I (DNase I) transfected into COS-cells: its distribution during apoptotic cell death.

COS-cells were transiently transfected with the pSG5 plasmid containing the cDNA of rat parotid deoxyribonuclease I (DNase I) either in right or inverse orientation. Expression of DNase I in transfected cells was only observed when the plasmid contained the cDNA in the right orientation. Expression of DNase I was monitored by measuring the DNase I specific DNA-degrading activity present in the conditioned cell culture medium and in cell homogenates. The expressed DNase I activity could be inhibited by monospecific polyclonal antibodies and by G-actin. Immunofluorescence indicated that approximately 20% of the COS-cells transfected with the DNase I-cDNA in right orientation expressed DNase I. These transfected cells contained large amounts of DNase I, which was found to be localized within the rough endoplasmic reticulum, the Golgi-complex and finally concentrated in a perinuclear location. Occasionally cells were observed which contained the DNase I in small apparently secretory transport vesicles. Transfected cells with perinuclear concentration of DNase I exhibited progressive nuclear destruction, i.e., pyknosis and cytoplasmic shrinkage. Solely the DNA extracted from isolated nuclei of cells transfected with the DNase I-cDNA in correct orientation revealed an internucleosomal DNA-degradation (ladder formation) typical for apoptosis after incubation in the presence of CaCl2 and MgCl2. Only the conditioned medium of COS-cells transfected with the right-oriented DNase I-cDNA contained the nucleolytic activity able to internucleosomally degrade the chromatin of substrate nuclei. Thus, these results indicate that overexpression of DNase I alone is sufficient to induce the morphological and biochemical changes observed during apoptosis.

Animals↗
PubMed1992

Functional characterisation of serum DNase I in MRL-lpr/lpr mice.

The autosomal defect in Fas antigen leads CD4-CD8-T-cells to accumulate in lymph nodes and spleen of MRL-lpr/lpr mice. MRL-lpr/lpr mice present increased levels of DNase I as compared to the control strain MRL-+/+. This DNase I, which most probably originates from the accumulated CD4-CD8-T-cells, cleaves nuclear DNA with a strong preference for internucleosomal sites yielding, in the presence of both Ca2+ and Mg2+, a pattern of fragments typical for apoptosis. Furthermore, we show that this "apoptosis-ladder" can be obtained with purified DNase I in presence of normal serum.

Animals↗
PubMed1992

Propidium iodide staining correlates with the extent of DNA degradation in isolated nuclei.

Gradual degradation of internucleosomal DNA is a hallmark of apoptosis and can be simulated by incubating isolated thymocyte nuclei in the presence of 5 mM Mg2+ and 5 mM Ca2+ at 37 degrees C. Staining of nuclei with the DNA binding fluorescent dye propidium iodide (PI) showed that intensity of fluorescence correlated with the extent of DNA degradation. PI fluorescence was increased in the presence of DNase I. Thus it seems that the cleavage of chromatin DNA by DNase 1 or by the endogenous enzyme increases the accessibility of DNA for the dye. No increase of fluorescence was observed in the presence of the known inhibitors of the endogenous endonuclease: Zn2+ and EGTA. However, the presence of Zn2+ led to decreased staining of the nuclei by PI and caused a shift in the scatter profile of the nuclei, suggesting that a conformational change of chromatin is induced by this ion. This correlation between intensity of PI staining and DNA degradation should be useful to compare endogenous nuclease levels in lymphocyte populations.

Animals↗
PubMed1991

Clusterin (complement lysis inhibitor) forms a high density lipoprotein complex with apolipoprotein A-I in human plasma.

Clusterin/human complement lysis inhibitor (CLI) is incorporated stoichiometrically into the soluble terminal complement complex and inhibits the cytolytic reaction of purified complement components C5b-9 in vitro. Using an anti-clusterin affinity column, we found that an additional protein component with a molecular mass of 28-kDa co-purifies with clusterin from human plasma. We show by immunoblotting and amino acid sequencing that this component is apolipoprotein A-I (apoA-I). By using physiological salt buffers containing 0.5% Triton X-100, apoA-I is completely dissociated from clusterin bound to the antibody column. Free clusterin immobilized on the antibody-Sepharose selectively retains apoA-I from total human plasma. Delipidated apoA-I and to a lesser extent ultracentrifugation-purified high density lipoproteins (HDL) adsorbed to nitrocellulose also have a binding affinity for purified clusterin devoid of apoA-I. The isolated apoA-I-clusterin complex contains approximately 22% (w/w) lipids which are composed of 54% (mole/mol) total cholesterol (molar ratio of unesterified/esterified cholesterol, 0.58), 42% phospholipids, and 4% triglycerides. In agreement with the low lipid content, apoA-I-clusterin complexes are detected only in trace amounts in HDL fractions prepared by density ultracentrifugation. In free flow isotachophoresis, the purified apoA-I-clusterin complex has the same mobility as the native clusterin complex in human plasma and is found in the slow-migrating HDL fraction of fasting plasma. Our data indicate that clusterin circulates in plasma as a HDL complex, which may serve not only as an inhibitor of the lytic terminal complement cascade, but also as a regulator of lipid transport and local lipid redistribution.

Apolipoprotein A-I↗
PubMed1991

Assembly of macromolecular pores by immune defense systems.

Immune defence systems (complement, cytolytic lymphocytes) make use of transmembrane pores assembled from up to 20 soluble monomers in a highly regulated process to induce cell death. Inhibitors of pore formation have been found which protect blood, endothelial and epithelial cells from the destructive effect of complement lesions. Recently, a pore-forming protein showing immunological crossreactivity to complement C9 has been found in the protozoan parasite Trypanosoma cruzi, thereby extending this protein family and generalizing its means of generating non-selective membrane permeability.

Animals↗