Joachim Stahl

Ribosomal Protein S24 Gene Is Mutated in Diamond-Blackfan Anemia

Diamond-Blackfan anemia (DBA) is a rare congenital red-cell aplasia characterized by anemia, bone-marrow erythroblastopenia, and congenital anomalies and is associated with heterozygous mutations in the ribosomal protein (RP) S19 gene (RPS19) in approximately 25% of probands. We report identification of de novo nonsense and splice-site mutations in another RP, RPS24 (encoded by RPS24 [10q22-q23]) in approximately 2% of RPS19 mutation-negative probands. This finding strongly suggests that DBA is a disorder of ribosome synthesis and that mutations in other RP or associated genes that lead to disrupted ribosomal biogenesis and/or function may also cause DBA.

Ribosomal Protein S7 Is Both a Regulator and a Substrate of MDM2

MDM2 associates with ribosomal protein S7, and this interaction is required to inhibit MDM2s E3 ligase activity, leading to stabilization of MDM2 and p53. Notably, the MDM2 homolog MDMX facilitates the inhibition of MDM2 E3 ligase activity by S7. Further, ablation of S7 inhibits MDM2 and p53 accumulation induced by different stress signals in some cell types. Thus, ribosomal/nucleolar stress is likely a key integrating event in DNA damage signaling to p53. Interestingly, S7 is itself a substrate for MDM2 E3 ligase activity both in vitro and in vivo. An S7-ubiquitin fusion protein (S7-Ub) selectively inhibits MDM2 degradation of p53 and is unaffected by MDMX. S7-Ub promotes apoptosis to a greater extent than S7 alone. This indicates that MDM2 ubiquitination of S7 is involved in sustaining the p53 response. Thus, S7 functions as both effector and affector of MDM2 to ensure a proper cellular response to different stress signals.

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.

Eukaryotic initiation factors eIF-2 and eIF-3: interactions, structure and localization in ribosomal initiation complexes

More than ten different protein factors are involved in initiation of protein synthesis in eukaryotes. For binding of initiator tRNA and mRNA to the 40S ribosomal subunit, the initiation factors eIF-2 and eIF-3 are particularly important. They consist of several different subunits and form stable complexes with the 40S ribosomal subunit. The location of eIF-2 and eIF-3 in these complexes as well as the interactions of the individual components have been analyzed by biochemical methods and electron microscopy. The results obtained are summarized in this article, and a model is derived describing the spatial arrangement of eIF-2 and eIF-3 together with initiator tRNA and mRNA on the 40S subunit. Conclusions on the location of functionally important sites of eukaryotic small ribosomal subunits are discussed with regard to the respective location of these sites in the prokaryotic counterpart.

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.

Human ribosomal protein S3a: cloning of the cDNA and primary structure of the protein.

The amino acid (aa) sequence of human ribosomal protein S3a (hRPS3a) was deduced partially from the nucleotide sequence of the corresponding cDNA and confirmed by direct aa sequencing from the N terminus of the purified hRPS3a protein. The cDNA clone was isolated from a cDNA expression library in the pEX vector using antibodies. The hRPS3a protein has 263 aa and its calculated M(r) is 29 813.

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.

The human ribosomal protein S7-encoding gene: isolation, structure and localization in 2p25

We have identified a gene encoding the human ribosomal protein (r-protein) S7. The S7 gene contains seven exons and six introns spanning about 6 kb. Organization of the gene is similar to that of Xenopus laevis S8, the only homologous intron-containing gene isolated so far. An mRNA transcribed from this gene has an open reading frame (ORF) of 582 nucleotides (nt), which encodes a protein of 194 amino acids (22.1 kDa). The transcription start point (tsp) was mapped by a primer extension assay to a C residue within a pyrimidine-rich tract. Human S7 (hS7) is identical to rat S7 (rS7) and exhibits significant similarity with the X. laevis, insect and plant homologs. We have used fluorescence in situ hybridization (FISH) to localize S7 to chromosome 2p25.

Cell-free phosphorylation of the murine small heat-shock protein hsp25 by an endogenous kinase from Ehrlich ascites tumor cells

The small heat-shock protein hsp25 of the Ehrlich ascites tumor exists in one non-phosphorylated (hsp25/1) and two phosphorylated (hsp25/2, hsp25/3) isoforms. In stationary phase tumor cells, a protein kinase activity was detected which phosphorylates hsp25/1, resulting in the formation of several phosphorylated hsp25 isoforms, including those occurring naturally in the tumor. Cell-free phosphorylation of hsp25 required Mg2+ and ATP and was independent of Ca2+, phosphatidylserine, cAMP and cGMP. Polymyxin B inhibited, specifically, hsp25 phosphorylation, whereas trifluoperazine, staurosporine and the protein inhibitor of protein kinase A had no effect. In its properties, the hsp25 phosphorylating kinase differs from other common kinases such as protein kinases A and C, calcium/calmodulin-dependent kinases, and the ribosomal protein S6 kinase.

Role of Small Heat Shock Proteins in the Cardiovascular System

There are five different small heat shock proteins (sHSPs), HSP27, B-crystallin, HSP20, HSP32 and ubiquitin, that occur at relatively high levels in heart tissues which therefore can be considered important for the cardiovascular system. The properties of the three structurally related sHSPs, HSP27, B-crystallin and HSP20, and their location in developing and differentiated heart tissues, in particular in cardiomyocytes, support their participation in heart muscle contraction, in which way, however, is in detail not yet elucidated. The functional role of these sHSPs is most likely influenced and regulated by phosphorylation via the MAPKAP kinase signalling system in correlation with stress-dependent protein kinases as well as by interactions with other important cardiac proteins as actin desmin, and vimentin. A fourth small heat shock protein, HSP32, is the enzyme hemoxygenase 1 (HO-1), part of the intracellular oxidoreductase system. It is induced in the cardiovascular system by various kinds of oxidative stress. High levels of HSP32 contribute to cardioprotection by maintaining and regulating the intracellular glutathione level. Finally, the role of the smallest heat shock protein, ubiquitin, and its possible importance for degradation and removal of molecularly damaged proteins is discussed in some detail. Altogether, these sHSPs are expressed in considerable amounts in heart tissues of many animals and elevated levels can be induced under stress and pathophysiological conditions. The cardioprotective functions of these proteins may contribute in different ways also to cardiac recovery from certain pathological states.