Heinz Bielka

Over-expression of the small heat-shock protein, hsp25, inhibits growth of Ehrlich ascites tumor cells

hsp25 is a small, growth‐related, mammalian stress protein which is highly accummulated in the stationary phase or Ehrlich ascites tumor in vivo. Ehrlich ascites cells cultivated in vitro under conditions of continuous exponential growth express hsp25 only at a low level. These cells were stably transfected with an eukaryotic expression vector carrying the coding sequence of the small heat‐shock protein, hsp25, under control of the murine metallothionein promoter. The resulting cell lines (EAT 116 and EAT 118) exhibit constitutive over‐expression of the small heat‐shock protein. hsp25, which can be further increased by induction with cadmium. Both cell lines show increased thermoresistance. The in vitro proliferation rate of the transfected cell lines EAT 116 and EAT 118 is significantly decreased depending on the degree of cadmium‐regulated over‐expression of hsp25. Furthermore, a significant delay in Ehrlich ascites tumor growth in mice using the hsp25 over‐expressing cells for primary inoculation could be demonstrated.

Crosslinking of initiation factor eIF-2 to proteins of the small subunit of rat liver ribosomes.

Eukaryotic mltiation factor eIF-2 binds Met-tRNA, and GTP to 40 S ribosomal subunits [l-7] . The bmdmg site for eIF-2 on the 40 S subunit was studied with 14C-labeled eIF-2 and ‘H-labeled ribosomal subunits, using DBI as a crosslinking reagent. High molecular weight protein conjugates were isolated which predominantly contained ribosomal protems S2, S3 and S18 (according to [ 1 l] ) covalently bound to the mitiation factor.

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.

Growth phase-dependent proteins of the Ehrlich ascites tumor analyzed by one- and two-dimensional electrophoresis

The decrease of the growth rate of the Ehrlich ascites tumor is correlated with changes in the de novo synthesis and abundance of several proteins as is shown by electrophoretic methods. The synthesis of a number of these growth-dependent proteins can be induced in vitro by incubation of the tumor cells in the presence of serum.

Cisplatin induces the small heat shock protein HSP25 and thermotolerance in Ehrlich ascites tumor cells

Exposure of Ehrlich ascites tumor (EAT) cells to the anticancer drug cisplatin results in an elevated abundance of three isoforms of the small heat shock protein hsp25 without inducing the general stress response as commonly observed after heat shock. The most effective cisplatin concentration (2.5 microM) is also most efficient in arresting cells in S phase suggesting a relationship between hsp25 expression and cell cycle events. Exposure to cisplatin results also in an increased thermotolerance of EAT cells.

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.

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.

Laser Raman studies of rat liver ribosomal 5 S RNA

The secondary and tertiary structure of ribosomal 5 S RNAs has been studied using a variety of physical and chemical techniques (review [ 11). As part of our spectroscopic investigations on ribosomal RNAs we report here on Raman spectroscopic data of the native form of 5 S RNA of rat liver ribosomes. Laser Raman spectroscopy has proved to be a sensitive probe for the structure of RNAs, especially for the determination of the type and extent of base stacking, the amount of order in the ribophosphate backbone and the percentage of U residues in base-paired regions [2-51. Raman data have been published for E. coli 5 S RNA [5] and yeast 5 S RNA [6]. Our Raman data for rat liver 5 S RNA indicate a very high regularity of the ribophosphate backbone in the native form of rat liver 5 S RNA, comparable to native tRNAs. The stacking efficiency of the guanine ba.ses is much higher in 5 S RNA than in yeast tRNAPhe, while that of the adenine bases is lower. The results support a base pairing pattern [7] which is very similar to that proposed in [8].