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Biomedical subjects

L Kuehn

Publications and source records attributed to L Kuehn.

At least 19 recordsLinked to original sources
PubMed2001

Hormone-triggered conformational changes within the insulin-receptor ectodomain: requirement for transmembrane anchors.

Interaction between two alphabeta half-receptors within the (alphabeta)(2) holoreceptor complex is required for insulin binding with high affinity and for insulin-triggered changes of size and shape. To understand the underlying structure-function relationship, two truncated receptor constructs have been characterized. Reduction in the Stokes radius and increase in the sedimentation coefficient, which are characteristic for wild-type receptors, were entirely lacking for the recombinant human insulin receptor (HIR) ectodomain (HIR-ED). Stokes radii of about 5.8 nm and sedimentation coefficients of 10.2 S were found for both insulin-bound and free HIR-EDs. However, attaching the membrane anchors to the ectodomain, as with the recombinant membrane-anchored ectodomain (HIR-MAED) construct, was sufficient to restore not only high-affinity hormone binding but also the marked insulin-inducible alterations in hydrodynamic properties. The Stokes radii of HIR-MAED complexes, as assessed by non-denaturing PAGE, decreased upon insulin binding from 9.5 nm to 7.9 nm. In parallel, the sedimentation coefficient was increased from 9.0 S to 9.8 S. CD and fluorescence spectroscopy of HIR-MAED revealed only minor insulin-induced changes in the secondary structure. Similarity with wild-type receptors has also been demonstrated by the differential insertion of insulin-bound and free HIR-MAED complexes into artificial bilayer membranes of Triton X-114. The results are consistent with a model of receptor function that ensures a global insulin-triggered reorientation of subdomains within the ectodomain moieties while the secondary structure is essentially retained. For the rearrangement of such subdomains, the transmembrane anchors confer essential structural constraints on the receptor ectodomain.

Chromatography, Gel↗
PubMed2001

Reconstitution of hybrid proteasomes from purified PA700-20 S complexes and PA28alphabeta activator: ultrastructure and peptidase activities.

The activity of the proteasome, the major non-lysosomal proteinase in eukaryotes, is stimulated by two activator complexes, PA700 and PA28. PA700-20 S-PA700 proteasome complexes, generally designated as 26 S proteasomes, degrade proteins, whereas complexes of the type PA28-20 S-PA28 degrade only peptides. We report, for the first time, the in vitro reconstitution of previously identified hybrid proteasomes (PA700-20 S-PA28) from purified PA700-20 S proteasome complexes and PA28 activator. In electron micrographs, the hybrid appears as a corkscrew-shaped particle with a PA700 and a PA28 activator each bound to a terminal alpha-disk of the 20 S core proteasome. The multiple peptidase activities of hybrid proteasomes are not different from those of PA28-20 S-PA28 or PA700-20 S-PA700 complexes.

Amino Acid Sequence↗
PubMed2001

Pronounced up-regulation of the PA28alpha/beta proteasome regulator but little increase in the steady-state content of immunoproteasome during dendritic cell maturation.

Dendritic cells (DC) are professional antigen-presenting cells that activate CTL by presenting MHC class I-restricted peptides that are processed through the proteasome pathway. Previously, we reported that upon DC maturation the synthesis is switched towards the exclusive production of immunoproteasomes containing the active site subunits LMP2, LMP7 and MECL-1. In this study we investigated the mechanism by which proteasome assembly is regulated in mature DC. Quantitative analysis of mRNA expression showed very limited transcriptional induction of LMP7, MECL-1 and UMP1 in mature DC and a moderate mRNA increment for LMP2 and PA28alpha and beta. We investigated a role of PA28alpha/beta in regulating proteasome assembly in DC. PA28alpha/beta coprecipitated with 13S/16S proteasome precursor complexes but associated with mature constitutive and immunoproteasomes to the same extent. Furthermore, we determined the steady-state proteasome subunit composition in DC. Replacement of constitutive proteasomes by immunoproteasomes in maturing DC was very slow and occurred only to a minor extent. Our data suggest that the limited turnover of 20S proteasomes in mature DC probably contributes little to recently reported marked differences in antigen presentation between immature and mature DC and that alternative mechanisms may be responsible for this phenomenon.

Binding Sites↗
PubMed2000

Different proteasome subtypes in a single tissue exhibit different enzymatic properties.

It is concluded from many experiments that mammalian tissues and cells must contain a heterogeneous population of 20 S proteasome complexes. We describe the purification and separation by chromatographic procedures of constitutive 20 S proteasomes, 20 S immuno-proteasomes and intermediate-type 20 S proteasomes from a given tissue. Our data demonstrate that each of these three groups comprises more than one subtype and that the relative ratios of the subtypes differ between different rat tissues. Thus, six subtypes could be identified in rat muscle tissue. Subtypes I and II are constitutive proteasomes, while subtypes V and VI comprise immuno-proteasomes. Subtypes III and IV belong to a group of intermediate-type proteasomes. The subtypes differ with regard to their enzymatic characteristics. Subtypes I-III exhibit high chymotrypsin-like activity and high peptidylglutamylpeptide hydrolysing activity, while these activities are depressed in subtypes IV-VI. In contrast, trypsin-like activity of subtypes IV-VI is enhanced in comparison to subtypes I-III. Importantly, the subtypes also differ in their preferential cleavage site usage when tested by digestion of a synthetic 25mer polypeptide substrate. Therefore, the characteristics of proteasomes purified from tissues or cells represent the average of the different subtype activities which in turn may have different functions in vivo.

Amino Acid Sequence↗
PubMed1997

Structural and functional properties of proteasome activator PA28.

The proteasome activator PA28 or 11S regulator is a protein complex composed of two different but homologous polypeptides, termed PA28alpha and PA28beta. The purified activator protein (approximately 200 kDa) is a ring-shaped heteromultimer containing the two polypeptides, possibly with an (alpha3beta3 stoichiometry. The activator, which by itself shows no hydrolytic activity elicits activation of the proteasome's multiple peptidase activities by binding to the terminal rings of the proteinase. In vitro, active PA28 can be reconstituted from isolated alpha and beta subunits, yielding two different oligomers: with the single alpha subunit, PA28alpha homomultimers with moderate stimulatory activity toward 20S proteasomes are obtained whereas isolated beta-subunits are unable to form oligomers and are devoid of stimulatory activity. However, in the presence of both subunits, alphabeta heteromultimers form, concomitant with restoration of full stimulatory activity. The recent finding that PA28 modulates the proteasome-catalyzed production of antigenic peptides presented to the immune system on MHC class I molecules indicates a cellular function of the activator in antigen processing.

Animals↗
PubMed1997

Using nursing standards to evaluate competency.

A competency evaluation program uses nursing standards to link performance management, quality improvement and education to patient care outcomes. This program enables nurse managers to capitalize on the data already available to them and helps meet the Joint Commission on Accreditation of Healthcare Organizations standards. An orientation competency assessment and an annual competency evaluation are provided.

Accreditation↗
PubMed1996

Reconstitution of proteasome activator PA28 from isolated subunits: optimal activity is associated with an alpha,beta-heteromultimer.

PA28, a 200 kDa activator of 20S proteasomes, was purified from human placenta and was gel electrophoretically resolved into two different subunits, alpha and beta. In reconstitution experiments, alpha-subunits alone were found to re-associate forming homooligomers with an M(r) of about 200 kDa, which elicit a stimulatory effect on proteasomal peptide-hydrolyzing activity, albeit at a moderate level. Under the same conditions, isolated beta-subunits were neither found to associate nor did they display stimulatory activity. Significantly, when both alpha- and beta-subunits were present in the reconstitution assay, heteromultimers formed, concomitant with a marked increase in stimulatory activity when compared with that of alpha-homooligomers. The reconstituted PA28alpha,beta protein is indistinguishable from purified PA28 by several criteria: it displays the same molecular mass, shows the same abundance of alpha- and beta-subunits and has a similar stimulatory activity toward 20S proteasomes. These results indicate that optimal PA28 activity is associated with a heteromultimeric structure which contains the alpha- and beta-subunits in fixed stoichiometry, most likely as an alpha3beta3-heterohexamer.

Blotting, Western↗
PubMed1996

Coordinated dual cleavages induced by the proteasome regulator PA28 lead to dominant MHC ligands.

The eukaryotic 20S proteasome is known to associate with the IFN gamma-inducible regulator PA28. We analyzed the kinetics of product generation by 20S proteasomes with and without PA28. In the absence of PA28, the 20S proteasome rapidly generates peptides that have been cleaved only once, while internal fragments accumulate only slowly. In the presence of PA28, products generated by two flanking cleavages appear immediately as main products while the generation of single-cleavage products is strongly reduced. Kinetic data support a PA28-induced, coordinated double-cleavage mechanism. In particular, degradation of peptides derived from mouse cytomegalovirus pp89 and JAK1 kinase in the presence of PA28 leads to strongly enhanced production of the respective major histocompatibility complex ligands and potential precursors. These results show that PA28 profoundly alters the cleavage mechanism of the proteasome and appears to optimize the generation of dominant T-cell epitopes.

Amino Acid Sequence↗
PubMed1996

A role for the proteasome regulator PA28alpha in antigen presentation.

Cytotoxic T cells recognize viral proteins as peptide fragments which are produced in the cytosol and transported on major histocompatibility complex (MHC) class I proteins to the cell surface. Viral peptides that meet the stringent binding characteristics of class I proteins are generated by the 20S proteasome. The interferon (IFN)-gamma-inducible activator of the 20S proteasome, PA28, strongly influences the proteasomal cleavage pattern in vitro. This led us to investigate whether changes in cellular levels of PA28 affect the efficiency of viral antigen processing. A mouse fibroblast line expressing the murine cytomegalovirus pp89 protein was transfected with either the human or murine gene encoding the PA28alpha subunit, which is sufficient to activate the peptide-hydrolysing activity of the 20S proteasome in vitro. Here we report that enhanced expression of PA28alpha at a level similar to that obtained after IFN-gamma induction resulted in a marked enhancement of recognition by pp89-specific cytotoxic T cells; the presentation of influenza nucleoprotein was also significantly improved. These results demonstrate a fundamental in vivo function for PA28alpha in antigen processing.

Amino Acid Sequence↗
PubMed1996

Proteasome activator PA28 and its interaction with 20 S proteasomes.

An activator of the 20 S proteasome has been purified to apparent homogeneity from rabbit erythrocytes, liver, and skeletal muscle. The activator displays an M(r) of about 200,000 upon sizing chromatography and, as judged by gel electrophoresis under denaturing conditions, is composed of two species of subunit of about equal abundance and with M(r) of 31 and 29 kDa. Upon isoelectric focusing, the activator is resolved into two major bands with pI values in the range of pH 5.1 and 5.5, corresponding to the two subunits. Limited proteolytic cleavage with trypsin results, for each subunit, in a distinct fragmentation pattern, indicating that in the rabbit, the native activator molecule occurs either as two homomultimers or as heteromultimers. The activator shows no hydrolytic activity by itself. However, when combined with proteasomes, it enhances, in a dose-related manner, the distinct peptidase activities of the proteinase. The activation process requires binding of the activator protein to the proteinase. This association, however, is reversible with recovery of active proteinase and activator protein. In vitro experiments suggest that, in vivo, the activator is bound to 20 S proteasomes rather than occurring as the free molecule.

Animals↗
PubMed1995

The interferon-gamma-inducible 11 S regulator (PA28) and the LMP2/LMP7 subunits govern the peptide production by the 20 S proteasome in vitro.

Antigenic peptides presented on major histocompatibility complex (MHC) class I molecules to cytotoxic T cells are generated in the cytosol by the 20 S proteasome. Upon stimulation of antigen presenting cells with interferon-gamma, two constitutive subunits of the 20 S proteasome are replaced by the MHC-encoded subunits low molecular mass polypeptide (LMP) 2 and LMP 7. In addition the expression of the two subunits of the 11 S regulator of the 20 S proteasome (PA28) are increased. As the function of LMP2 and LMP7 in antigen presentation is still controversial, we tested whether these subunits might operate by modifying proteasome activation through the 11 S regulator. We strongly overexpressed the two LMP subunits separately or together by transfection in murine fibroblasts. Isolated 20 S proteasomes from LMP transfectants were applied in digests of a 25-mer peptide in the presence or absence of a purified preparation of 11 S regulator from rabbit erythrocytes. Analysis of the cleavage products by high performance liquid chromatography and electrospray mass spectroscopy revealed marked differences in the peptide product profile in dependence on the LMP2 and LMP7 content. While the 11 S regulator did not preferentially activate LMP2 or 7 containing proteasomes, the binding of the 11 S regulator to any of the proteasome preparations markedly changed both the quality and quantity of peptides produced. These results suggest that the 11 S regulator increases the spectrum of peptides which can be generated in antigen presenting cells.

Amino Acid Sequence↗
PubMed1995

Studies on the activation by ATP of the 26 S proteasome complex from rat skeletal muscle.

The 26 S proteasome complex is thought to catalyse the breakdown of ubiquitinated proteins within eukaryotic cells. In addition it has been found that the complex also degrades short-lived proteins such as ornithine decarboxylase in a ubiquitin-independent manner. Both proteolytic processes are paralleled by the hydrolysis of ATP. Here we show that ATP also affects the hydrolytic activity towards fluorigenic peptide substrates by the 26 S proteasome complex from rat skeletal muscle tissue. Low concentrations of ATP (about 25 microM) optimally activate the so-called chymotryptic and tryptic activity by increasing the rate of peptide hydrolysis but not peptidylglutamylpeptide hydrolysis. Activation of the enzyme by ATP is transient but this effect can be enhanced and prolonged by including in the assay an ATP-regenerating system, indicating that ATP is hydrolysed by the 26 S proteasome complex. Although ATP cannot be substituted for by adenosine 5'-[beta,gamma-methylene]triphosphate or AMP, hydrolysis of the phosphoanhydride bond of ATP seems not to be necessary for the activation process of the proteasome complex, a conclusion drawn from the findings that ATP analogues such as adenosine 5'-[beta,gamma-imido]triphosphate, adenosine 5'-O-[gamma-thio]triphosphate, adenosine 5'-O-[beta-thio]-diphosphate and adenosine 5'-[alpha,beta-methylene]triphosphate give the same effect as ATP, and vanadate does not prevent ATP activation. These effects are independent of the presence of Mg2+. Thus, ATP and other nucleotides may act as allosteric activators of peptide-hydrolysing activities of the 26 S proteasome complex as has also been found with the lon protease from Escherichia coli.

Adenosine Triphosphate↗
PubMed1995

The 20S/26S proteasomal pathway of protein degradation in muscle tissue.

Similar to all other eukaryotic cells and tissues muscle tissue contains the proteolytic system of 20S/26S proteasomes with the 20S proteasome existing predominantly in a latent state. Unlike with the mammalian enzyme in vitro transition from the latent to the activated state of the 20S proteasomes isolated from muscle of several fish species and from lobster can be achieved by heat shock. It is very likely that the activated state of the 20S proteasome corresponds to the physiologically active form of the enzyme since only that one is able to attack sarcoplasmic and myofibrillar proteins to any significant extent. As perfusion of rat hindquarters with presumptive low molecular mass activators like free fatty acids does not result in an activation of the muscle proteasome other--possibly protein activators--may serve this purpose in vivo. The 26S proteasome complex may be regarded as such a proteasome/activator complex. The 26S proteasome complex has the ability to degrade protein (-ubiquitin-conjugates) by an ATP-consuming reaction. Since increased amounts of ubiquitinated proteins as well as an enhanced activity of the ATP (-ubiquitin)-dependent proteolytic system have been measured in rat muscle tissue during various catabolic conditions, it is not unlikely that this pathway is responsible for catalysis of muscle protein breakdown.

Adenosine Triphosphate↗
PubMed1993

In vitro activation of the 20S proteasome.

The effect of chemical compounds like sodium dodecyl sulfate (SDS), fatty acid esters of glycerol, carnitine and coenzyme A, phospholipids, histones, polylysines as well as homobifunctional chemical cross-linkers on the various proteolytic activities of mammalian proteasomes have been tested. Most of the reagents enhance these activities, and some, e.g. fatty acid CoA esters, histones and the chemical cross-linkers, exert dual effects, i.e. activation and inhibition at the same time, depending on the activity measured. With optimally activating concentrations of SDS, no structural changes in proteasomes can be detected by electron microscopy. Formation of micelles at supra-optimal detergent concentrations may be a reason for irreversible denaturation of the proteasome.

Amino Acid Sequence↗
PubMed1992

Biochemical properties of the proteasome from Thermoplasma acidophilum.

We have purified proteasomes to apparent homogeneity from the archaebacterium Thermoplasma acidophilum. This proteinase has a molecular mass of about 650 kDa and an isoelectric point of 5.6. The proteasome hydrolyses peptide substrates containing an aromatic residue adjacent to the reporter group, as well as [14C]methylated casein optimally at pH 8.5 and 90 degrees C. The enzyme activity is enhanced severalfold by Mg2+ and Ca2+ at 25-500 mM. This increase in activity results primarily from a change in Km. The serine-proteinase inhibitors diisopropylfluorophosphate and 3,4-dichloroisocoumarin irreversibly inhibit the enzyme, obviously by modification of both the alpha and beta subunits in the proteasome. The inhibition of proteasomal activity by the peptidylchloromethanes, Cbz-Leu-Leu-CH2Cl and Cbz-Ala-Ala-Phe-CH2Cl (Cbz, benzyloxycarbonyl), is reversible and predominantly of a competitive type. The enzyme is not activated by any of the compounds that typically stimulate the activities of the eukaryotic proteasome.

Amino Acid Sequence↗