Gudrun Lutsch

Smooth-muscle contraction without smooth-muscle myosin

Here we have used gene-targeting to eliminate expression of smooth-muscle myosin heavy chain. Elimination of this gene does not affect expression of non-muscle myosin heavy chain, and knockout individuals typically survive for three days. Prolonged activation, by KCl depolarisation, of intact bladder preparations from wild-type neonatal mice produces an initial transient state (phase 1) of high force generation and maximal shortening velocity, which is followed by a sustained state (phase 2) characterized by low force generation and maximal shortening velocity. Similar preparations from knockout neonatal mice do not undergo phase 1, but exhibit a normal phase 2. We propose that, in neonatal smooth muscle phase 1 is generated by recruitment of smooth-muscle myosin heavy chain, whereas phase 2 can be generated by activation of non-muscle myosin heavy chain. We conclude that phase 1 becomes indispensable for survival and normal growth soon after birth, particularly for functions such as homeostasis and circulation.

Abundance and Location of the Small Heat Shock Proteins HSP25 and αB-Crystallin in Rat and Human Heart

BACKGROUND In the heart, there are high constitutive levels of the two related small heat shock proteins, HSP25 and alphaB-crystallin. To gain insight into their functional role, we have analyzed abundance and location of both proteins in rat and human hearts at different stages of development and in diseased state. METHODS AND RESULTS Immunoblotting analysis of rat ventricular tissue at fetal, neonatal, and adult stages reveals the level of HSP25 to decline strongly during development, whereas the level of alphaB-crystallin remains nearly constant. In parallel, the portion of phosphorylated isoforms of HSP25 decreases as shown by two-dimensional polyacrylamide gel electrophoresis. HSP25 is detected in cardiomyocytes and endothelial and vascular smooth muscle cells, whereas alphaB-crystallin is detected in cardiomyocytes only by immunofluorescence and immunoelectron microscopy. Both proteins colocalize in the I-band and M-line region of myofibrils in cardiomyocytes. In diseased and transplanted adult human hearts, HSP25 and alphaB-crystallin levels are considerably elevated compared with fetal hearts. In failing adult human hearts, phosphorylated isoforms of HSP25 predominate, and cardiomyocytes with a partial dislocation of HSP25 and alphaB-crystallin are observed. CONCLUSIONS Differential accumulation and location of HSP25 and alphaB-crystallin in heart tissue during development imply distinct functions of both proteins, which seem to be involved in organization of cytoskeletal structures. As judged by level, phosphorylation state, and location of both small heat shock proteins, diseased adult human hearts share features with fetal hearts.

Signaling from β-adrenoceptor to L-type calcium channel: identification of a novel cardiac protein kinase A target possessing similarities to AHNAK

A novel calcium channel‐associated protein of ~700 kDa has been identified in mammalian cardiomyocytes that undergoes substantial cAMP‐dependent protein kinase (PKA) phosphorylation. It was therefore designated as phosphoprotein 700 (pp700). The pp700 interacts specifically with the β2 subunit of cardiac L‐type calcium channels as revealed by coprecipitation experiments using affinity‐purified antibodies against different calcium channel subunits. It is surprising that amino acid sequence analysis of pig pp700 revealed homology to AHNAK‐encoded protein, which was originally identified in human cell lines of neural crest origin as 700‐kDa phosphoprotein. Cardiac AHNAK expression was assessed on mRNA level by reverse transcriptase‐polymerase chain reaction. Sequence‐directed antibodies raised against human AHNAK recognized pp700 in immunoblotting and immunoprecipitation experiments, confirming the homology between both proteins. Anti‐AHNAK antibodies labeled preferentially the plasma membrane of cardiomyocytes in cryosections of rat cardiac tissue and isolated cardiomyocytes. Sarcolemmal pp700/ AHNAK localization was not influenced by stimulation of either the PKA or the protein kinase C pathway. In back‐phosphorylation studies with cardiac biopsies, we identified distinct pp700 pools. The membrane‐associated fraction of pp700 underwent substantial in vivo phosphorylation on β‐adrenergic receptor stimulation by isoproterenol, whereas the cytoplasmic fraction of pp700 was not accessible to endogenous PKA. It is important that in vivo phosphorylation occurred in that pp700 fraction which coprecipitated with the calcium channel β subunit. We hypothesize that both phosphorylation of pp700 and its coupling to the β subunit play a physiological role in cardiac β ‐adrenergic signal transduction. Haase, H., Podzuweit, T., Lutsch, G., Hohaus, A., Kostka, S., Lindschau, C., Kott, M., Kraft, R., Morano, I. Signaling from β‐adrenoceptor to L‐type calcium channel: identification of a novel cardiac protein kinase A target that has similarities to AHNAK. FASEB J. 13, 2161–2172 (1999)

Interaction of huntingtin fragments with brain membranes--clues to early dysfunction in Huntington's disease.

Huntingtin is a large, multi‐domain protein of unknown function in the brain. An abnormally elongated polyglutamine stretch in its N‐terminus causes Huntingtons disease (HD), a progressive neurodegenerative disorder. Huntingtin has been proposed to play a functional role in membrane trafficking via proteins involved in endo‐ and exocytosis. Here, we supply evidence for a direct association between huntingtin and membranes. In the brains of R6/2 mice with HD pathology, a 64 kDa N‐terminal huntingtin fragment accumulated in postsynaptic membranes during the pre‐symptomatic period of 4–8 weeks of age. In addition, an oligomeric fragment of approximately 200 kDa was detected at 8 weeks of age. Simultaneous progressive changes in distribution of amphiphysin, synaptojanin, and subunits of NMDA‐ and AMPA‐receptors provide a strong indication of dysfunctional synaptic trafficking. Composition of the major phospholipids in the synaptic membranes was unaffected. In vitro, large unilamellar vesicles of brain lipids readily associated with soluble N‐terminal huntingtin exon 1 fragments and stimulated fibrillogenesis of mutant huntingtin aggregates. Moreover, interaction of both mutant and wild‐type huntingtin exon 1 fragments with brain lipids caused bilayer perturbation, mediated through a proline‐rich region adjacent to the polyglutamines. This suggests that lipid interactions in vivo could influence misfolding of huntingtin and may play an early role in HD pathogenesis.

Supramolecular structure of the recombinant murine small heat shock protein hsp25

The size and shape of the recombinant murine small heat shock protein, hsp25, have been analyzed by hydrodynamic and electron microscopic methods. According to these studies recombinant hsp25 exists in large complexes with a sphere‐like shape and diameters of 15–18 nm. The molecular mass of these complexes amounts to about 730 kDa indicating that they are composed of about 32 monomers.

Immunochemical detection of proteins in the small subunit of rat liver ribosomes involved in binding of the ternary initiation complex

An essential step of peptide chain initiation in eukaryotes is the binding of the ternary initiation complex [Met-tRNAfX eIF-2 X GTP] to the P site of small ribosomal subunit, thus forming the quaternary initiation complex [40 S subunit X Met-tRNA,X eIF-2 X GTP] [ l-41. First attempts to analyze proteins of the small subunit of rat liver ribosomes involved in binding of components of the ternary initiation complex have been made by the application of antibodies [5] and by crosslinking experiments [6]. In [S] we described the effect of antibodies against ribosomal protein S3 (according to the newly proposed common nomenclature of eukaryotic ribosomal proteins [7]) on the binding of [3H]Met-tRNAfin complex with eIF-2 and GTP to the 40 S ribosomal subunit. We now report further experiments with antibodies against 9 proteins of the small ribosomal subunit. From the strong inhibitory activity of the antibodies against ribosomal proteins S3, S6, and S13 and their location on the small ribosomal subunit as studied by immune electron microscopy ([8,9] and unpublished) it is concluded that these proteins are involved in the P site organization and that the P site is located in the head region of the small ribosomal subunit.

The carboxyl-terminal ahnak domain induces actin bundling and stabilizes muscle contraction

Ahnak, a 700 kDa protein, is expressed in a variety of cells and has been implicated in different cell‐type‐specific functions. In the human heart, we observed an endogenous carboxyl‐terminal 72 kDa ahnak fragment that copurified with myofibrillar proteins. Immunocytochemistry combined with confocal microscopy localized this fragment to the intercalated discs and close to the Z‐line of cardiomyocytes. No endogenous myofibrillar ahnak fragment was observed in the skeletal muscle. We elucidated the role of the recombinant carboxyl‐terminal ahnak fragment (ahnak‐C2) in actin filament organization and in the function of muscle fibers. Addition of ahnak‐C2 to actin filaments induced filament bundling into paracrystalline‐like structures as revealed by electron microscopy. Incubation of demembranated skeletal muscle fibers with ahnak‐C2 attenuated the decline in isometric force development upon repeated contraction–relaxation cycles. Our results suggest that the carboxyl‐terminal ahnak domain exerts a stabilizing effect on muscle contractility via its interaction with actin of thin filaments.

Identification of proteins of the 40 S ribosomal subunit involved in interaction with initiation factor eIF‐2 in the quaternary initiation complex by means of monospecific antibodies

Monospecific polyclonal antibodies against seven proteins of the 40 S subunit of rat liver ribosomes were used to identify ribosomal proteins involved in interaction with initiation factor eIF‐2 in the quaternary initiation complex [eIF‐2 × GMPPCP × [3H]Met‐tRNAf × 40 S ribosomal subunit]. Dimeric immune complexes of 40 S subunits mediated by antibodies against ribosomal proteins S3a, S13/16, S19 and S24 were found to be unable to bind the ternary initiation complex [eIF‐2 × GMPPCP × [3H]Met‐tRNAf]. In contrast, 40 S dimers mediated by antibodies against proteins S2, S3 and S17 were found to bind the ternary complex. Therefore, from the ribosomal proteins tested, only proteins S3a, S13/16, S19 and S24 are concluded to be involved in eIF‐2 binding to the 40 S subunit.

Localization of rat liver ribosomal protein S2 and its involvement in initiation factor eIF-2 binding to the 40 S ribosomal subunit

The elucidation of the localization and arrangement of RNA and proteins and their function in active sites of ribosomes, and thus the understanding of the molecular events which occur during protein synthesis, need the application of different experimental techniques [ 1,2] . In the following the location of ribosomal protein S2 in the small subunit of rat liver ribosomes using immunoelectron microscopy is shown. Furthermore, from inhibition experiments with antibodies against protein S2 it is demonstrated that S2 is involved in binding of the eukaryotic initiation factor IF-2 to the small ribosomal subunit. From the results it can be concluded that protein S2 is located at or near the ribosomal P-site and that the P-site is organized at least partially in the so-called head region of the small ribosomal subunit.

Increased level of HSP27 but not of HSP72 in human heart allografts in relation to acute rejection.

Background. Increased expression of heat shock proteins (HSPs) was assumed during cardiac allograft rejection. To find evidence for this in man, we quantified HSP27 and HSP72 in cardiac allograft biopsies. Methods. In parallel to histological assessment of rejection, HSP27 was quantified by Western blotting in a total of 43 biopsies sampled from 3 patients. HSP72 was analyzed in parallel in 30 of the 43 cases. For comparison, HSPs were analyzed in myocardium. Results. HSP27 was significantly higher in rejecting cardiac allografts than in non-rejecting allografts and non-failing myocardium (1.52±0.25 vs. 0.83±0.11 vs. 0.50±0.05 &mgr;g/mg protein). Similarity for HSP72 (6.27±1.54 vs. 4.06±1.03 vs. 6.27±0.76 &mgr;g/mg protein) was not found. Conclusion. For the first time in humans with cardiac allograft rejection, increased expression of HSP27, which could be important for cardiac self-protection, was demonstrated. For the lack of increased HSP72 expression, the influence of the cyclosporine A treatment was discussed.