YB-1 is a DNA/RNA-binding nucleocytoplasmic shuttling protein whose regulatory effect on many DNA-and RNAdependent events is determined by its localization in the cell. Distribution of YB-1 between the nucleus and the cytoplasm is known to be dependent on nuclear targeting and cytoplasmic retention signals located within the C-terminal portion of YB-1. Here, we report that YB-1 undergoes a specific proteolytic cleavage by the 20S proteasome, which splits off the C-terminal 105-amino-acidlong YB-1 fragment containing a cytoplasmic retention signal. Cleavage of YB-1 by the 20S proteasome in vitro appears to be ubiquitin-and ATP-independent, and is abolished by the association of YB-1 with messenger RNA. We also found that genotoxic stress triggers a proteasome-mediated cleavage of YB-1 in vivo and leads to accumulation of the truncated protein in nuclei of stressed cells. Endoproteolytic activity of the proteasome may therefore play an important role in regulating YB-1 functioning, especially under certain stress conditions.

Surface labeling of Escherichia coli ribosomes with the use of the tritium bombardment technique has revealed a minor unidentified ribosome-bound protein (spot Y) that is hidden in the 70S ribosome and becomes highly labeled on dissociation of the 70S ribosome into subunits. In the present work, the N-terminal sequence of the protein Y was determined and its gene was identified as yfia, an ORF located upstream the phe operon of E. coli. This 12.7-kDa protein was isolated and characterized. An affinity of the purified protein Y for the 30S subunit, but not for the 50S ribosomal subunit, was shown. The protein proved to be exposed on the surface of the 30S subunit. The attachment of the 50S subunit resulted in hiding the protein Y, thus suggesting the protein location at the subunit interface in the 70S ribosome. The protein was shown to stabilize ribosomes against dissociation. The possible role of the protein Y as ribosome association factor in translation is discussed.ribosomal proteins ͉ ribosome dissociation ͉ ribosomal surface ͉ tritium labeling T he hot tritium bombardment technique is based on replacement of hydrogen by tritium in covalent bonds of thin surface layer in macromolecules (1). The technique appears to be the most direct approach for studies of protein topography on surfaces of biological structures. Protein exposure on the surfaces of viruses (1-3), membranes (4), and ribosomes (5-9) has been determined by the use of this technique.Studying proteins of the ribosome surface with this method has lead to the finding that unidentified minor component of the ribosome corresponding to spot Y on the ribosomal protein map becomes highly exposed on dissociation of the 70S ribosome into subunits (5, 7, 9). The reassociation of ribosomal subunits by increasing Mg 2ϩ concentration resulted in reshielding of the protein Y, suggesting its location at the ribosomal subunit interface (9). In this study, the protein was identified and isolated. Its interface location was confirmed by experiments on hot tritium bombardment. The protein was shown to support the 70S ribosome in associated state at low concentration of Mg 2ϩ . Materials and MethodsMaterials. Buffer reagents were obtained from Sigma, acrylamide and methylenebisacrilamide were from Fluka, urea was from Bio-Rad, DNase I was from Serva, Butyl-Toyopearl 650S from Toyo Soda (Tokyo), and DEAE-Sepharose Fast Flow from Pharmacia. Sucrose, acetone, hydrogen peroxide, and acetic acid were from ReaKhim (Moscow, Russia).Preparation of 70S Ribosomes and Total Ribosomal Protein. Ribosomes were prepared from Escherichia coli MRE-600 cells according to Staehelin et al. (10) with modifications described in ref. 9. Salt-washed ribosomes used in binding assay, as well as ribosomal subunits, were prepared as described by Gavrilova et al. (11) with substitution of pelleting by ultracentrifugation for precipitation with (NH 4 ) 2 SO 4 at the final stage of the procedure.The standard procedure of acetic acid extraction and precipitation with acetone (12) was used to p...

The plasmid-located gene cafl encoding the capsular antigen fraction I (Fl) of Yersiniu pestis was cloned and sequenced. The gene codes for a 170 amino acid peptide with a deduced M, of 17.6 kDa. The signal peptide sequence was highly homologous to the E. co/i consensus signal sequence. The Fl was assumed to have p-sheet structure for the most part. The region located between amino acids 100 and 150 was suggested to contain putative antigenic determinants and to stimulate T cells. 1, INTRODUCTIONThe ultimate goal of infectious disease research is their prevention. Vaccination is one of the most effective ways in which that goal can be attained. It is necessary to know the gene structure and putative immunogenic surface structures of antigens to create recombinant vaccines.More than 10 antigens have been isolated from Yersinia pestis. The capsular antigen fraction 1 (Fl) was shown to be a highly protective antigen among such thermolabile antigens as D, Fl, T, V and W [I). Some properties of the Fl structure have been studied recently [2], but the nucleotide and amino acid sequences have been unknown so far. Here, we report the cloning and sequencing of the Y. pestis cafl gene coding for the Fl , and the predicted secondary structure with potential antigenic determinants. MATERIALS AND METHODS Bacterial strains, plasmids and DNA manipulafionsThe E. co/i strains LE392 and HBIOI were used as transistent hosts for cosmid pHC79 [3], and pUC18 or f9 [4], respectively. Y. pestisF1 positive vaccine strain EV was obtained from the Culture Collection, All-Union Antiplague Institute 'Microb', USSR. Cultures were grown overnight while shaking at 37°C in liquid LB or on solid medium supplemented with the relevant antibiotics for plasmid selection. Construction of a gene library, screening, and subcloningThe Y. pestis plasmid pFra DNA (about 110 kb in size) was partially digested with EcoRI, ligated with EeoRIdigested cosmid pHC79 and packaged in vitro. The library was amplified in E. coli LE392 and ApRTcR colonies selected were further screened for Fl production by enzyme immunoassay. The isolated cosmid ~153 containing a 40-kb fragment of pFra DNA was then digested with EcoRl and an 8.6kb fragment was cloned into pHC79. The resulting cosmid pFS2 was digested with SalI and Hind111 and a 4.5kb fragment was cloned into pUCI9. The plasmid pFS2-13 generated was used for gene sequencing. The 1 .O-kb Alul fragment of pFS2-I 3 was cloned into the SmaIdigested pUCl8 (plasmid pF18L) and sequenced. Protein sequencingThe Fl protein was isolated from culture medium and purified by polyacrylamide gel electrophoresis as described [2]. The N-terminus of the mature protein was identified by a PTH-amino acid analyzer (Model 120A, Applied Biosystems). Secondary structure and anrigenic determinant analysis

Scattered literature data on biologically active hemoglobin-derived peptides are collected in the form of tables. Respective structure-functional correlations are analyzed and the general conclusion is reached that hemoglobin fragments must have a profound physiological function. Evidence is presented that generation of hemoglobin fragments starts inside the erythrocytes. At that stage alpha- and beta-globin chains of hemoglobin predominantly give rise to relatively long peptides containing ca. 30 amino acid residues. The primary proteolysis is followed by the next degradation step coupled with excretion of newly formed shorter peptides form red blood cells. Both the primary and the secondary proteolysis products are subjected to further stepwise C- and N-terminal chain shortening, giving rise to families of closely related peptides that are actually found in animal tissue extracts. The possible sites of primary proteolysis are compared with the positions of the exposed secondary structure elements within the monomeric alpha- and beta-globins as well as the tetrameric hemoglobin. Two tentative schemes are proposed for hemoglobin degradation, one of which starts at the globin loops exposed on the surface of the tetramer and the other, at monomeric globins where more sites are available for the action of proteases. The concept of a "tissue-specific peptide pool" is formulated, describing a novel system of peptidergic regulation, complementary to the conventional hormonal and neuromodulatory systems. According to that description, hemoglobin is only a single example, although an important one, of a vast number of functional proteins providing their proteolytically derived fragments for maintaining the tissue homeostasis.