Apoptosis-induced cleavage of keratin 15 and keratin 17 in a human breast epithelial cell line.

Keratin 15 (K15) and keratin 17 (K17) are intermediate filament (IF) type I proteins that are responsible for the mechanical integrity of epithelial cells. By analyzing the human breast epithelial cell line H184A1 before and after induction of apoptosis by high-resolution two-dimensional gel electrophoresis (2-DE) we identified the caspase-mediated cleavage of keratins 15 and 17. After induction of apoptosis three fragments of both K15 and K17 could be observed by 2 -DE. K15 and K17 proteolysis was observed during staurosporine-induced apoptosis and anoikis (anchorage-dependent apoptosis) as well and was shown to be caspase-dependent. By using mass spectrometry we could determine the caspase cleavage sites, one in K15 and two in K17. The sequence VEMD/A at the cleavage site located in the conserved linker region was found in K15 and K17. A further cleavage site was identified in the tail region of K17 with the recognition motif EVQD/G.

A direct photo-activated affinity modification of tetracycline transcription repressor protein TetR(D) with tetracycline(1).

Results of a first successful application of a direct photo-induced affinity modification of Tet repressor (TetR(D)) protein with tetracycline within a complex of known three-dimensional structure are described. The conditions of the modification have provided suitable yields of the modified complex and allowed characterization of the modified segments of the protein. The potential of tetracycline as a fine modifying reagent was established. In the complex of TetR(D) protein with tetracycline, the antibiotic modifies at least two segments, Ile59-Glu73 and Ala173-Glu183, which form a binding tunnel for the drug according to the X-ray analysis. These data open possibilities for the use of different tetracycline targets for structural studies in solution.

New approaches for innovations in sensitive Edman sequence analysis by design of a wafer-based chip sequencer.

In the last few years the development of new mass spectrometric techniques enabled fast and sensitive protein analysis by the introduction of mass spectrometry (MS) fingerprinting and MS/MS sequencing. For these methods mixtures of peptide fragments of the proteins can be employed, whereas the Edman degradation method requests purified peptides. On the other hand, Edman sequencing has the advantages that interpretation of the data is more simple, extended sequences can be derived, and reliable sequence information on unknown proteins is possible. Hence, Edman chemistry as an alternative technique to MS is still valuable. But higher sensitivity of the sequencers is needed in order to meet modern demands, e.g. in proteomics research. We designed a wafer-based chip sequencer for protein and peptide sequencing in the femto- to attomole range. The main advantage of our new design is the complete integration of dead volume free valves together with reactor and converter and volume-measuring loops within one wafer-based system. In this system aggressive chemicals and solvents for the Edman degradation can be delivered in sub-microliter amounts, which allows a considerable shortening of the degradation cycles. Further, we developed sensitive maintenance and tightness tests to prove a precise and reproducible delivery of the chemicals and the reduction of drying times as compared with conventional sequencers. Real parallel processing of several samples can easily be implemented. The system is designed to serve future needs in protein research.

Mammalian mitochondrial ribosomal proteins (4). Amino acid sequencing, characterization, and identification of corresponding gene sequences.

Mitochondrial ribosomal proteins (MRPs) are required for the translation of all 13 mitochondrial encoded genes in humans. It has been speculated that mutations and polymorphisms in the human MRPs may be a primary cause of some oxidative phosphorylation disorders or modulate the severity and tissue specificity of pathogenic mitochondrial DNA mutations. Although the sequences of most of the yeast MRPs are known, only very few mammalian and nearly no human MRPs have been completely characterized. MRPs differ greatly in sequence, and sometimes biochemical properties, between different species, not allowing easy recognition by sequence homology. Therefore, the Mammalian Mitochondrial Ribosomal Consortium is using a direct approach of purifying individual mammalian (bovine) MRPs, determining their N-terminal and/or internal peptide sequences using different protein sequencing techniques, and using the resulting sequence information for screening expressed sequence tags and genomic data bases to determine human, mouse, and rat homologues of the bovine proteins. Two proteins of the large and three proteins of the small ribosomal subunit have been analyzed in this manner. Three of them represent "new," i.e. formerly unknown mammalian mitochondrial ribosomal protein classes. Only one of these three different MRPs shows significant sequence similarities to known ribosomal proteins. In one case, the corresponding human genomic DNA sequences were found in the data bases, and the exon/intron structure was determined.

Heart-specific splice-variant of a human mitochondrial ribosomal protein (mRNA processing; tissue specific splicing).

It has been proposed that splice-variants of proteins involved in mitochondrial RNA processing and translation may be involved in the tissue specificity of mitochondrial DNA disease mutations (Fischel-Ghodsian, 1998. Mol. Genet. Metab. 65, 97-104). To identify and characterize the structural components of mitochondrial RNA processing and translation, the Mammalian Mitochondrial Ribosomal Consortium has been formed. The 338 amino acid (aa) residues long MRP-L5 was identified (O'Brien et al., 1999. J. Biol. Chem. 274, 36043-36051), and its transcript was screened for tissue specific splice-variants. Screening of the EST databases revealed a single putative splice-variant, due to the insertion of an exon consisting of 89 nucleotides prior to the last exon. Screening of multiple cDNA libraries revealed this inserted exon to be present only in heart tissue, in addition to the predominant MRP-L5 transcript. Sequencing of this region confirmed the EST sequence, and showed in the splice-variant a termination triplet at the beginning of the last exon. Thus the inserted exon replaces the coding sequence of the regular last exon, and creates a new 353 aa long protein (MRP-L5V1). Sequence analysis and 3D modeling reveal similarity between MRP-L5 and threonyl-t-RNA synthetases, and a likely RNA binding site within MRP-L5, with the C-terminus in proximity to the RNA binding site. Sequence analysis of MRP-L5V1 also suggests a likely transmembrane domain at the C-terminus. Thus it is possible that the MRP-L5V1 C-terminus could interfere with RNA binding and may have gained a transmembrane domain. Further studies will be required to elucidate the functional significance of MRP-L5V1.

Mammalian mitochondrial ribosomal proteins (2). Amino acid sequencing, characterization, and identification of corresponding gene sequences.

Four different classes of mammalian mitochondrial ribosomal proteins were identified and characterized. Mature proteins were purified from bovine liver and subjected to N-terminal or matrix-assisted laser-desorption mass spectroscopic amino acid sequencing after tryptic in-gel digestion and high pressure liquid chromatography separation of the resulting peptides. Peptide sequences obtained were used to virtually screen expressed sequence tag data bases from human, mouse, and rat. Consensus cDNAs were assembled in silico from various expressed sequence tag sequences identified. Deduced mammalian protein sequences were characterized and compared with ribosomal protein sequences of Escherichia coli and yeast mitochondria. Significant sequence similarities to ribosomal proteins of other sources were detected for three out of four different mammalian protein classes determined. However, the sequence conservation between mitochondrial ribosomal proteins of mammalian and yeast origin is much less than the sequence conservation between cytoplasmic ribosomal proteins of the same species. In particular, this is shown for the mammalian counterparts of the E. coli EcoL2 ribosomal protein (MRP-L14), that do not conserve the specific and functional highly important His(229) residue of E. coli and the corresponding yeast mitochondrial Rml2p.

Nucleoid proteins of pea chloroplasts: detection of a protein homologous to ribosomal protein.

Basic proteins were isolated from purified pea chloroplast nucleoids by acid extraction. Using RP-HPLC, the component composition of the basic proteins was studied. SDS-PAGE of major HPLC-fractions showed that the basic nucleoid proteins are heterogeneous with mol. masses of components from 17 to 30 kDa. One polypeptide with mol. mass of 28 kDa (P28) was obtained by RP-HPLC. The sequencing of three tryptic peptides of P28 (T6, T17, and T19) showed that they are homologous to the ribosomal protein L19 of Saccharomyces cerevisiae. The possible functional role of ribosomal proteins in chloroplast nucleoids is discussed.

Preparative high-resolution two-dimensional electrophoresis enables the identification of RNA polymerase B transcription factor 3 as an apoptosis-associated protein in the human BL60-2 Burkitt lymphoma cell line.

Apoptosis or programmed cell death is essential in the process of controlling lymphocyte growth and selection. We identified RNA polymerase B transcription factor 3 (BTF3), which is associated with anti-IgM antibody-mediated apoptosis, using a subclone of the human Burkitt lymphoma cell line BL60. To identify the transcription factor BTF3, which is expressed only in minor amounts, we used preparative high-resolution two-dimensional gel electrophoresis (2DE) employing carrier ampholytes for isoelectric focusing. Comparison of the 2DE protein patterns from apoptotic and nonapoptotic cells showed BTF3 as a predominantly altered protein spot. The characterization of the differentially expressed transcription factor and 13 marker proteins described in this study were performed by internal Edman microsequencing and/or by peptide mass fingerprinting using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The proteome analysis was significantly improved by performing the newly developed preparative high-resolution two-dimensional gels employing high protein concentrations.

Study of Burkitt lymphoma cell line proteins by high resolution two-dimensional gel electrophoresis and nanoelectrospray mass spectrometry.

We paper describe a mass spectrometric approach generally applicable for the rapid identification and characterization of proteins isolated by two-dimensional gel electrophoresis (2-DE). The highly sensitive nanoflow-electrospray mass spectrometry employing a quadrupole-time of flight mass spectrometer was used for the direct identification of proteins from the peptide mixture generated from only one high resolution 2-DE gel without high performance liquid chromatography (HPLC) separation or Edman sequencing. Due to the high sensitivity and high mass accuracy of the instrument employed, this technique proved to be a powerful tool for the identification of proteins from femtomole amounts of materials. We applied the technique for the investigation of Burkitt lymphoma BL60 cell proteins. This cell line has been used as a model to assign apoptosis-associated proteins by subtractive analysis of normal and apoptotic cells. From the nuclear fraction of these cells, 36 protein spots were examined, from only one micropreparative Coomassie Brilliant Blue R-250 stained gel, after proteolytic digestion by matrix assisted laser desorption ionization (MALDI) and nanospray mass spectrometry (MS). In combination with database searches, of 33 proteins were successfully identified by nanospray-MS/MS-sequencing of up to eight peptides per protein. Three proteins were new proteins not listed in any of the available databases. Some of the identified proteins are known to be involved in apoptosis processes, the others were common proteins in the eukaryotic cell. The given technique and the protein data are the basis for construction of a database to compare normal and apoptosis-induced cells and, further, to enable fast screening of drug impact in apoptosis-associated processes.

Mammalian mitochondrial ribosomal proteins. N-terminal amino acid sequencing, characterization, and identification of corresponding gene sequences.

The integrity of healthy mitochondria is supposed to depend largely on proper mitochondrial protein biosynthesis. Mitochondrial ribosomal proteins (MRPs) are directly involved in this process. To identify mammalian mitochondrial ribosomal proteins and their corresponding genes, we purified mature rat MRPs and determined 12 different N-terminal amino acid sequences. Using this peptide information, data banks were screened for corresponding DNA sequences to identify the genes or to establish consensus cDNAs and to characterize the deduced MRP open reading frames. Eight different groups of corresponding mammalian MRPs constituted from human, mouse, and rat origin were identified. Five of them show significant sequence similarities to bacterial and/or yeast mitochondrial ribosomal proteins. However, MRPs are much less conserved in respect to the amino acid sequence among species than cytoplasmic ribosomal proteins of eukaryotes and bacteria.

Identification of apoptosis-associated proteins in a human Burkitt lymphoma cell line. Cleavage of heterogeneous nuclear ribonucleoprotein A1 by caspase 3.

Apoptosis or programmed cell death is essential in the process of controlling lymphocyte growth and selection. We identified proteins that are involved in anti-IgM antibody-mediated apoptosis using a subclone of the human Burkitt lymphoma cell line BL60. Apoptosis-associated proteins were detected by high resolution two-dimensional gel electrophoresis on a micropreparative scale. Comparison of the high resolution two-dimensional gel electrophoresis protein patterns from apoptotic and non-apoptotic cells showed differences in approximately 80 spots including protein modifications. Analysis of the predominantly altered proteins was performed by internal Edman microsequencing and/or by peptide mass fingerprinting using matrix-assisted laser desorption/ionization mass spectrometry. Analysis was significantly improved by using new micropreparative high resolution two-dimensional gels employing high protein concentrations. The following 12 apoptosis-associated proteins were identified: heterogeneous nuclear ribonucleoprotein (hnRNP) A1, hnRNP C1/C2, FUSE-binding protein, dUTPase, lymphocyte-specific protein LSP1, UV excision repair protein RAD23 homologue B (HHR23B), 60 S acidic ribosomal protein P0 (L10E), heterochromatin protein 1 homologue alpha (HP1alpha), nucleolin, lamin, neutral calponin, and actin. Fragmentation of actin, hnRNP A1, hnRNP C1/C2, 60 S acidic ribosomal protein P0, lamin, and nucleolin could be inhibited by benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethyl ketone, a selective irreversible inhibitor of CPP32 (caspase 3).

Precise determination of RNA-protein contact sites in the 50 S ribosomal subunit of Escherichia coli.

RNA-protein cross-linked complexes were isolated and purified to obtain precise data about RNA-protein contact sites in the 50 S ribosomal subunit of Escherichia coli. N-terminal microsequencing and matrix-assisted laser desorption ionization MS were used to identify the cross-linking sites at the amino acid and nucleotide levels. In this manner the following contact sites of five ribosomal proteins with the 23 S rRNA were established: Lys-67 of L2 to U-1963, Tyr-35 of L4 to U-615, Lys-97 of L21 to U-546, Lys-49 of L23 to U-139 or C-140 and Lys-71 and Lys-74 of L27 to U-2334.

Functional implications of ribosomal protein L2 in protein biosynthesis as shown by in vivo replacement studies.

The translational apparatus is a highly complex structure containing three to four RNA molecules and more than 50 different proteins. In recent years considerable evidence has accumulated to indicate that the RNA participates intensively in the catalysis of peptide-bond formation, whereas a direct involvement of the ribosomal proteins has yet to be demonstrated. Here we report the functional and structural conservation of a peptidyltransferase centre protein in all three phylogenetic domains. In vivo replacement studies show that the Escherichia coli L2 protein can be replaced by its homologous proteins from human and archaebacterial ribosomes. These hybrid ribosomes are active in protein biosynthesis, as proven by polysome analysis and poly(U)-dependent polyphenylalanine synthesis. Furthermore, we demonstrate that a specific, highly conserved, histidine residue in the C-terminal region of L2 is essential for the function of the translational apparatus. Replacement of this histidine residue in the human and archaebacterial proteins by glycine, arginine or alanine had no effect on ribosome assembly, but strongly reduced the translational activity of ribosomes containing these mutants.

Mitochondrial ribosomal proteins (MRPs) of yeast.

Mitochondrial ribosomal proteins (MRPs) are the counterparts in that organelle of the cytoplasmic ribosomal proteins in the host. Although the MRPs fulfil similar functions in protein biosynthesis, they are distinct in number, features and primary structures from the latter. Most progress in the eludication of the properties of individual MRPs, and in the characterization of the corresponding genes, has been made in baker's yeast (Saccharomyces cerevisiae). To date, 50 different MRPs have been determined, although biochemical data and mutational analysis propose a total number which is substantially higher. Surprisingly, only a minority of the MRPs that have been characterized show significant sequence similarities to known ribosomal proteins from other sources, thus limiting the deduction of their functions by simple comparison of amino acid sequences. Further, individual MRPs have been characterized functionally by mutational studies, and the regulation of expression of MRP genes has been described. The interaction of the mitochondrial ribosomes with transcription factors specific for individual mitochondrial mRNAs, and the communication between mitochondria and the nucleus for the co-ordinated expression of ribosomal constituents, are other aspects of current MRP research. Although the mitochondrial translational system is still far from being described completely, the yeast MRP system serves as a model for other organisms, including that of humans.

Contact sites of peptide-oligoribonucleotide cross-links identified by a combination of peptide and nucleotide sequencing with MALDI MS.

We have investigated peptide-oligoribonucleotide complexes isolated from cross-linked Escherichia coli 30S ribosomal subunits in order to identify the contact sites of these complexes at the molecular level. For this purpose, reversed-phase (RP) HPLC-purified peptide-oligoribonucleotide complexes were submitted to N-terminal amino acid sequencing in order to determine the cross-linked peptide moiety and were analyzed using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for calculation of the nucleotide composition of the cross-linked complex. Subsequently, for nucleotide sequence information the complexes were partially hydrolyzed or treated with exonucleases and analyzed again by MALDI-MS. Applying this technique, we were able to identify the cross-linked oligoribonucleotide parts in contact with distinct peptide regions derived from ribosomal proteins S4, S7, S8, and S17 from E. coli.

The modular organization of multifunctional peptide synthetases.

Gramicidin S synthetase 2 from B. brevis was affinity labeled at its valine thiolation center with the thiol reagent N-[3H]ethylmaleimide. From a tryptic digest of the enzyme-inhibitor complex a radioactive fragment was isolated in pure form by two reversed-phase HPLC steps. It was identified by liquid-phase N-terminal sequencing in combination with electrospray mass spectrometry (ESI-MS) as a hexadecapeptide containing the thiolation motif LGG(H/D)S(L/I). By ESI-MS it was demonstrated that a 4'-phosphopantetheine cofactor was attached to this fragment at its reactive serine. These results are consistent with the "Multiple Carrier Model" of nonribosomal peptide biosynthesis. Site-specific mutagenesis has been performed in thiolation, elongation, and epimerization motifs of some of the modules of surfactin synthetase from B. subtilis to clarify the function of prominent conserved amino acid residues in the intermediate steps of peptide biosynthesis. The modular structure of multifunctional peptide synthetases is discussed.