The small heat shock proteins (sHsps) from human (Hsp27) and mouse (Hsp25) form large oligomers which can act as molecular chaperones in vitro and protect cells from heat shock and oxidative stress when overexpressed. In addition, mammalian sHsps are rapidly phosphorylated by MAPKAP kinase 2/3 at two or three serine residues in response to various extracellular stresses. Here we analyze the effect of sHsp phosphorylation on its quaternary structure, chaperone function, and protection against oxidative stress. We show that in vitro phosphorylation of recombinant sHsp as well as molecular mimicry of Hsp27 phosphorylation lead to a significant decrease of the oligomeric size. We demonstrate that both phosphorylated sHsps and the triple mutant Hsp27-S15D,S78D,S82D show significantly decreased abilities to act as molecular chaperones suppressing thermal denaturation and facilitating refolding of citrate synthase in vitro. In parallel, Hsp27 and its mutants were analyzed for their ability to confer resistance against oxidative stress when overexpressed in L929 and 13.S.1.24 cells. While wild type Hsp27 confers resistance, the triple mutant S15D,S78D,S82D cannot protect against oxidative stress effectively. These data indicate that large oligomers of sHsps are necessary for chaperone action and resistance against oxidative stress whereas phosphorylation down-regulates these activities by dissociation of sHsp complexes to tetramers. Small heat shock proteins (sHsps)1 are constitutively expressed in virtually all organisms and exhibit a monomeric molecular mass of 15-42 kDa (for a recent review see Ref. 1). Within the cell they can form oligomeric complexes of up to 1 MDa (2). Overexpression of different mammalian sHsps increases cellular thermoresistance to a significant degree (3, 4). sHsps can, furthermore, function in different, seemingly unrelated processes like RNA stabilization (5), interaction with the cytoskeleton (6, 7), or apoptosis (8, 9). In vitro sHsps act as molecular chaperones preventing unfolded proteins from irreversible aggregation (10 -12) and, in cooperation with other factors, e.g. Hsp70 and ATP, facilitating productive refolding of unfolded proteins (13,14).In mammalian cells certain sHsps, e.g. mouse Hsp25 or human Hsp27, form a converging element of the cellular stress response since they show both a stress-induced increase in expression and phosphorylation. Under heat shock conditions increased phosphorylation can be detected after several minutes while changes in expression are detected after several hours (15). The rapid stress-induced phosphorylation is the result of stimulation of the p38 MAP kinase cascade and subsequent activation of MAPKAP kinases 2 and 3 which directly phosphorylate mammalian sHsps (16, 17) at several distinct sites (18,19). Since sHsp phosphorylation and stress-induced expression show different kinetics, it is assumed that phosphorylation of the pre-existing constitutively expressed sHsps is a first phase of the stress response while the elevated expression at a time w...

Alternative splicing of Wt1 results in the insertion or omission of the three amino acids KTS between zinc fingers 3 and 4. In vitro experiments suggest distinct molecular functions for + and -KTS isoforms. We have generated mouse strains in which specific isoforms have been removed. Heterozygous mice with a reduction of +KTS levels develop glomerulosclerosis and represent a model for Frasier syndrome. Homozygous mutants of both strains die after birth due to kidney defects. Strikingly, mice lacking +KTS isoforms show a complete XY sex reversal due to a dramatic reduction of Sry expression levels. Our data demonstrate distinct functions for the two splice variants and place the +KTS variants as important regulators for Sry in the sex determination pathway.

No abstract

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.

We have investigated the conformational transition and aggregation process of recombinant Syrian hamster prion protein (SHaPrP 90 -232 ) by Fourier transform infrared spectroscopy, circular dichroism spectroscopy, light scattering, and electron microscopy under equilibrium and kinetic conditions. SHaPrP 90 -232 showed an infrared absorbance spectrum typical of proteins with a predominant ␣-helical structure both at pH 7.0 and at pH 4.2 in the absence of guanidine hydrochloride. At pH 4.2 and destabilizing conditions (0.3-2 M guanidine hydrochloride), the secondary structure of SHaPrP 90 -232 was transformed to a strongly hydrogen-bonded, most probably intermolecularly arranged antiparallel ␤-sheet structure as indicated by dominant amide I band components at 1620 and 1691 cm ؊1 . Kinetic analysis of the transition process showed that the decrease in ␣-helical structures and the increase in ␤-sheet structures occurred concomitantly according to a bimolecular reaction. However, the concentration dependence of the corresponding rate constant pointed to an apparent third order reaction. No ␤-sheet structure was formed within the dead time (190 ms) of the infrared experiments. Light scattering measurements revealed that the structural transition of SHaPrP 90 -232 was accompanied by formation of oligomers, whose size was linearly dependent on protein concentration. Extrapolation to zero protein concentration yielded octamers as the smallest oligomers, which are considered as "critical oligomers." The small oligomers showed spherical and annular shapes in electron micrographs. Critical oligomers seem to play a key role during the transition and aggregation process of SHaPrP 90 -232 . A new model for the structural transition and aggregation process of the prion protein is described.The prion protein (PrP) 1 is, following the protein-only hypothesis, the sole agent causing a group of neurodegenerative disorders (1, 2), the so-called prion diseases or prionoses (3). The most important ones among them are bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep, and Creutzfeldt-Jakob disease in humans.The crucial step in transmission and manifestation of prion diseases is the conversion of benign monomeric cellular prion protein (PrP C ), which has a mainly ␣-helical secondary structure, to pathogenic multimeric scrapie prion protein (PrP Sc ), which is predominantly folded into ␤-sheets (4, 5). It is noteworthy that PrP C and PrP Sc do not differ in their amino acid sequence.Similar mechanisms play an essential role in a number of other neurodegenerative disorders including Alzheimer's, Parkinson's, and Huntington's diseases. Therefore, the coupled processes of protein misfolding and aggregation, the kinetics of these processes, and the molecular species involved are of fundamental interest.Late products of the conversion are amyloid fibrils and amyloid plaques, which are widely considered to be direct effectors of the above mentioned disorders. However, evidence is accumulating that intermediates or by-product...