Spermidine synthase is prominently expressed in the striatal patch compartment and in putative interneurones of the matrix compartment.

The ubiquitous polyamines spermidine and spermine are known as modulators of glutamate receptors and inwardly rectifying potassium channels. They are synthesized by a set of specific enzymes in which spermidine synthase is the rate-limiting step catalysing the formation of the spermine precursor spermidine from putrescine. Spermidine and spermine were previously localized to astrocytes, probably reflecting storage rather than synthesis in these cells. In order to identify the cellular origin of spermidine and spermine synthesis in the brain, antibodies were raised against recombinant mouse spermidine synthase. As expected, strong spermidine synthase-like immunoreactivity was obtained in regions known to express high levels of spermidine and spermine, such as the hypothalamic paraventricular and supraoptic nuclei. In the striatum, spermidine synthase was found in neurones and the neuropil of the patch compartment (striosome) as defined by expression of the micro opiate receptor. The distinct expression pattern of spermidine synthase, however, only partially overlapped with the distribution of the products spermidine and spermine in the striatum. In addition, spermidine synthase-like immunoreactivity was seen in patch compartment-apposed putative interneurones. These spermidine synthase-positive neurones did not express any marker characteristic of the major striatal interneurone classes. The neuropil labelling in the patch compartment and in adjacent putative interneurones may indicate a role for polyamines in intercompartmental signalling in the striatum.

Defined sequence segments of the small heat shock proteins HSP25 and alphaB-crystallin inhibit actin polymerization.

The interaction of small heat shock proteins (sHSPs) with the actin cytoskeleton has been described and some members of this family, e.g. chicken and murine HSP25 (HSP27), inhibit the polymerization of actin in vitro. To analyse the molecular basis of this interaction, we synthesized a set of overlapping peptides covering the complete sequence of murine HSP25 and tested the effect of these peptides on actin polymerization in vitro by fluorescence spectroscopy and electron microscopy. Two peptides comprising the sequences W43 to R57 (peptide 6) and I92 to N106 (peptide 11) of HSP25 were found to be potent inhibitors of actin polymerization. Phosphorylation of N-terminally extended peptide 11 at serine residues known to be phosphorylated in vivo resulted in decline of their inhibitory activity. Interestingly, peptides derived from the homologous peptide 11 sequence of murine alphaB-crystallin showed the same behaviour. The results suggest that both HSP25 and alphaB-crystallin have the potential to inhibit actin polymerization and that this activity is regulated by phosphorylation.

Conversion of yeast phosphoglycerate kinase into amyloid-like structure.

Yeast phosphoglycerate kinase is a structurally well-characterized enzyme consisting of 415 amino acids without disulfide bonds. Anion-induced refolding from its acid-unfolded state gives rise to the formation of worm-like amyloid fibrils with a persistence length of 73 nm. Electron microscopy and small-angle X-ray scattering data indicate that the fibrils have an elliptical cross-section with dimensions of 10.2 nm x 5.1 nm. About half of all amino acids are organized in form of cross-beta structure which gives rise to typical infrared spectra, X-ray diffraction and yellow-green birefringence after Congo red staining. The kinetics of amyloid formation, monitored by infrared spectroscopy, dynamic light scattering and X-ray scattering, was found to be strongly dependent on protein concentration. The infrared data indicate that the formation of cross-beta structure practically comes to an end already after some hours, whereas the length-growth of the amyloid fibrils, monitored by small-angle X-ray scattering, was not yet completed after 1,300 hours.

Regulation of Hsp27 oligomerization, chaperone function, and protective activity against oxidative stress/tumor necrosis factor alpha by phosphorylation.

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.

The 80S rat liver ribosome at 25 A resolution by electron cryomicroscopy and angular reconstitution.

BACKGROUND: The ribosome is central to protein synthesis in all living organisms. Single-particle electron cryomicroscopy has recently led to the determination of three-dimensional structures of bacterial ribosomes to approximately 20 A, which have since revolutionised our understanding of ribosomal function. The structure we present here of the 80S rat liver ribosome leads the way to similar progress for mammalian ribosomes. RESULTS: Among the new details revealed by our 25 A structure of the 80S rat liver ribosome are channels within the subunits, a large 'flat ribosomal surface' (FRS) on the outer surface of the large subunit and structural extensions of the mammalian compared to the bacterial ribosome. The main large subunit channel in both the bacterial and the mammalian species starts at the peptidyl transferase centre, below the central protuberance, and ends in the FRS, at the lower back of the large subunit. Structurally, the channels of both species can be directly superimposed. CONCLUSIONS: The mammalian structural extensions--none of which trespass the FRS--can be interpreted in terms of rRNA inserts and additional protein content over that of bacterial ribosomes. The main large subunit channel, which ends at the FRS, is the best candidate for the exit channel for proteins targeted for the endoplasmic reticulum.

Abundance and location of the small heat shock proteins HSP25 and alphaB-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.

Phosphorylation and supramolecular organization of murine small heat shock protein HSP25 abolish its actin polymerization-inhibiting activity.

Characteristic features of mammalian small heat shock proteins are their rapid phosphorylation in response to stress and mitogenic signals and their ability to form multimeric particles of 200-700 kDa and large aggregates up to 5000 kDa. Recently, a chaperoning function and an actin polymerization-inhibiting activity were demonstrated for the recombinant murine and turkey small heat shock protein, respectively. In this paper, we demonstrate that the actin polymerization-inhibiting activity of the murine small heat shock protein HSP25 is dependent on the degree of its phosphorylation and structural organization. Non-phosphorylated and phosphorylated HSP25 monomers, as well as non-phosphorylated multimeric HSP25 particles, were isolated from Ehrlich ascites tumor cells by ammonium sulfate precipitation, column chromatography, and ultracentrifugation and tested for their actin polymerization-inhibiting activity. Fluorescence spectroscopy and electron microscopy were used to monitor actin polymerization. Non-phosphorylated HSP25 monomers were active in inhibiting actin polymerization with about 90% inhibition at a 1:1 ratio of actin to HSP25, while phosphorylated HSP25 monomers and non-phosphorylated multimeric HSP25 particles were inactive. Furthermore, we present electron microscopic data on the structure of HSP25 particles.