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Nanotransducers in cellular redox signaling: modification of thiols by reactive oxygen and nitrogen species.

The control of signal transduction involves post-translational modification of proteins at key amino acids. Cysteine residues are important in the control of 'redox' cell-signaling pathways, as thiol chemistry offers the possibility of modification by structurally diverse species, including those derived from oxidized lipids, peroxides or nitric oxide. An important and provocative study of the modification of thiols in the transcription factor OxyR recently extended this hypothesis. The findings offer the enticing possibility that the cell can distinguish between different degrees of oxidant and nitrosative exposure by modification at a single site on a signaling molecule.

Animals↗

Physiological role and regulation of the Na+/H+ exchanger.

In mammalian eukaryotic cells, the Na+/H+ exchanger is a family of membrane proteins that regulates ions fluxes across membranes. Plasma membrane isoforms of this protein extrude 1 intracellular proton in exchange for 1 extracellular sodium. The family of Na+/H+ exchangers (NHEs) consists of 9 known isoforms, NHE1-NHE9. The NHE1 isoform was the first discovered, is the best characterized, and exists on the plasma membrane of all mammalian cells. It contains an N-terminal 500 amino acid membrane domain that transports ions, plus a 315 amino acid C-terminal, the intracellular regulatory domain. The Na+/H+ exchanger is regulated by both post-translational modifications including protein kinase-mediated phosphorylation, plus by a number of regulatory-binding proteins including phosphatidylinositol-4,5-bisphosphate, calcineurin homologous protein, ezrin, radixin and moesin, calmodulin, carbonic anhydrase II, and tescalcin. The Na+/H+ exchanger is involved in a variety of complex physiological and pathological events that include regulation of intracellular pH, cell movement, heart disease, and cancer. This review summarizes recent advances in the understanding of the physiological role and regulation of this protein.

Allosteric Regulation↗

Establishing the yeast Saccharomyces cerevisiae as a system for expression of human proteins on a proteome-scale.

Structural genomics requires the application of a standardised process for overexpression of soluble proteins that allows high-throughput purification and analysis of protein products. We have developed a highly parallel approach to protein expression, including the simultaneous expression screening of a large number of cDNA clones in an appropriate vector system and the use of a protease-deficient host strain. A set of 221 human genes coding for proteins of various sizes with unknown structures was selected to evaluate the system. We transferred the cDNAs from an E. coli vector to the yeast expression vector by recombinational cloning, avoiding time-consuming recloning steps and the use of restriction enzymes in the cloning process. The subcloning yield was 95%, provided that a PCR fragment of the correct size could be obtained. Sixty percent of these proteins were expressed as soluble products at detectable levels and 48% were successfully purified under native conditions using the His6 tag fusion. The advantages of the developed yeast-based expression system are the ease of manipulation and cultivation of S. cerevisiae in the same way as with prokaryotic hosts and the ability to introduce post-translational modifications of proteins if required, thus being an attractive system for heterologous expression of mammalian proteins. The expression clones selected in this screening process are passed on to the fermentation process in order to provide milligram amounts of proteins for structure analysis within the 'Berlin Protein Structure Factory'. All data generated is stored in a relational database and is available on our website (http://www.proteinstrukturfabrik.de).

Aspartic Acid Endopeptidases↗

Quantitative and qualitative variation of ETS-1 transcripts in hematologic malignancies.

The ETS family proteins have a conserved DNA-binding domain and act as transcription factors. Three domains have been recently defined in human ETS-1 proteins and their role could depend upon the nature of alternative transcripts according to whether they possess or lack DNA binding and/or transcriptional activation domain and also point mutation that could affect these important domains. Expression of ETS-1 gene is very complex and is controlled at several levels: the initiation of transcription, alternative splicing, post-translational modification, and protein stability. As a selection apparently exists for ETS-1 gene activation in hematopoietic cells, we investigated a relation between quantitative and qualitative ETS-1 expression and leukemogenesis. Using Northern blot, polymerase chain reaction (PCR), and single strand conformation polymorphism (SSCP) methods, we analyzed quantitative and qualitative ETS-1 expression in a variety of hematological pathologies and cell lines of different origin. Two ETS-1 transcripts of 6.8 and 2.7 kb, resulting from differential polyadenylation site utilization and exhibiting different stability, were observed. We identified, in a great number of patients, the four alternative ETS-1 products, but the relative extent significance of the four transcripts was very different from one patient to another. A non-conservative mutation observed in one case of T-cell acute lymphoblastic leukemia (T-ALL) and in the ETS-1 transactivation domain raised the question of suppressor activity for some ETS-1 products, as it is now known that activators and repressors can be encoded by the same gene and consistently co-expressed in vivo.

Base Sequence↗

Role of phosphorylation in p53 acetylation and PAb421 epitope recognition in baculoviral and mammalian expressed proteins.

Post-translational modifications, such as phosphorylation and acetylation of the tumour suppressor protein p53, elicit important effects on the function and the stability of the resultant protein. However, as phosphorylation and acetylation are dynamic events subject to complex controls, elucidating the relationships between phosphorylation and acetylation is difficult. In the present study we sought to address this problem by comparing full-length wild-type p53 with full-length p53 proteins mutated at specific phosphorylation targets. Recombinant murine p53 proteins were expressed in insect cells (using the baculoviral expression vector system) and in a mammalian in vitro transcription/translation reticulocyte lysate system. In p53 proteins derived from baculoviral expression vectors, S37A (but not S37D) was found to abrogate phosphorylation at S15. Lysine 382 (K382) is constitutively acetylated and was shown to form part of the epitope recognized by PAb421. Lysine 373 (K373) was only acetylated following substitutions at S315 (S315A or S315D) or at S378 (S378A). Importantly, in baculoviral expressed proteins, PAb421 reactivity was independent of K373 acetylation status, indicating that acetylation at K382 specifically determines the PAb421 epitope.

Acetylation↗

Changes in protein synthetic activity in early Drosophila embryos mutant for the segmentation gene Krüppel.

We have identified early embryo proteins related to the segmentation gene Krüppel by [35S]methionine pulse labelling and two-dimensional gel electrophoresis. Protein synthesis differences shared by homozygous embryos of two Krüppel alleles when compared to heterozygous and wild-type embryos are reported. The study was extended to syncytial blastoderm stages by pulse labelling and gel analysis of single embryos, using Krüppel-specific proteins from gastrula stages as molecular markers for identifying homozygous Krüppel embryos. Localized expression of interesting proteins was examined in embryo fragments. The earliest differences detected at nuclear migration stages showed unregulated synthesis in mutant embryos of two proteins that have stage specific synthesis in normal embryos. At the cellular blastoderm stage one protein was not synthesized and two proteins showed apparent shifts in isoelectric point in mutant embryos. Differences observed in older embryos included additional proteins with shifted isoelectric points and a number of qualitative and quantitative changes in protein synthesis. Five of the proteins with altered rates of synthesis in mutant embryos showed localized synthesis in normal embryos. The early effects observed are consistent with the hypothesis that the Krüppel product can be a negative or positive regulator of expression of other loci, while blastoderm and gastrula stage shifts in isoelectric point indicate that a secondary effect of Krüppel function may involve post-translational modification of proteins.

Alleles↗

[Clinical applications of recombinant complement inhibitors].

The complement system constitutes an important part of the innate immune system. Complement activation leads to the generation of C3a, C4a and C5a anaphylatoxins and the membrane attack complex. The anaphylatoxins mediate multiple reactions in the acute inflammatory response. Membrane attack complex inserts molecules into target membranes and causes cell lysis. The complement system can not discriminate between self and non-self cells and the inappropriate complement activation may lead to host cell damage. This destructive activity is tightly regulated by family of structurally and functionally related soluble and membrane-bound proteins, which act as inhibitors of complement system. The inappropriate complement activation plays an essential role in the pathogenesis of many diseases. In the therapy of these diseases specific recombinant complement inhibitors can be used. Recombinant complement inhibitors can be produced in large amounts by different eukaryotic or prokaryotic systems. The choice of the system depends on kind of the post-translational modifications of proteins.

Anaphylatoxins↗

Microarray tools for deciphering complex diseases.

Individual genetic findings associated with complex diseases are unlikely to fully explain their substantial impact or provide new comprehensive insights into disease pathogenesis. These also lack the comprehensive data much needed for development of new effective drugs in majority of the disease cases in a population. In fact multilevel etiologic factors underlie almost all human diseases, including: environmental causes, epigenetic factors, DNA mutations, amplifications, and deletions, RNA expression levels, protein (translation, post translation modification, localization) and combinations thereof. Each individual might consist of different combinations of these multiple etiologic factors. Integrative evaluation of all these modifications will shed light on the whole identity of the disease and the underlying molecular mechanisms. Until now it was inconceivable to have a full grasp of such a complex etiology. Microarrays enable us to interrogate the individualized various factors (DNA, RNA and protein content) involved in disease state on genome-wide scale simultaneously and expeditiously in single cell or the tissue of interest (Figure 1). The new disciplines of microarray studies in combination hold the promise of effective, current, and comprehensive understanding of complex diseases and may be a good approach for reducing the costs and time lines associated with discovery and efficacy improvement of therapeutic drugs. In the future, through utilizing the colossal amount of microarray data findings, defining the structure, function, and dynamics of entire biological pathways and cellular networks under various physiological states, and the development of robust and efficient methods for analyzing and interpreting high dimensional data, it will be possible to connect combination of experimental results with individualized disease state. This will facilitate precise diagnosis prognosis and therapy.

Alternative Splicing↗

Mass spectrometric identification of proteins and characterization of their post-translational modifications in proteome analysis.

High-throughput DNA sequencing has resulted in increasing input in protein sequence databases. Today more than 20 genomes have been sequenced and many more will be completed in the near future, including the largest of them all, the human genome. Presently, sequence databases contain entries for more than 425.000 protein sequences. However, the cellular functions are determined by the set of proteins expressed in the cell--the proteome. Two-dimensional gel electrophoresis, mass spectrometry and bioinformatics have become important tools in correlating the proteome with the genome. The current dominant strategies for identification of proteins from gels based on peptide mass spectrometric fingerprinting and partial sequencing by mass spectrometry are described. After identification of the proteins the next challenge in proteome analysis is characterization of their post-translational modifications. The general problems associated with characterization of these directly from gel separated proteins are described and the current state of art for the determination of phosphorylation, glycosylation and proteolytic processing is illustrated.

Computational Biology↗

One-step purification of mammalian deoxyribonucleases I and differences among pancreas, parotid, and pancreas-parotid (mixed) types based on species- and organ-specific N-linked glycosylation.

Mammalian deoxyribonucleases I (DNase I) are classified into three types, namely, pancreas, parotid, and pancreas-parotid (mixed), based on differences in their tissue concentrations. In this study, DNase I purification by concanavalin A-wheat germ agglutinin mixture-agarose column from rat (parotid type), rabbit (mixed type), and pig (pancreas type) is described. This method permits a relatively easy one-step purification of DNase I from rat and rabbit parotid glands, the rat submaxillary gland, and porcine pancreas. To elucidate differences among the three types, these DNases I were subjected to enzymatic deglycosylation either by peptide N-glycosidase F (PNGase F) or endoglycosidase H (Endo H). Following deglycosylation, digests were separated on DNA-casting polyacrylamide gel electrophoresis. PNGase F produced a single lower mobility product in all samples. Endo H produced a double band in rat and rabbit parotid glands and porcine pancreas, and a single band in the rabbit pancreas corresponding with the PNGase F product. DNase I activity of the porcine pancreas was completely extinguished by deglycosylation, while that of the parotid glands and rabbit pancreas was unaffected. Our results suggest that the distinct properties of DNase I exhibited by the three types may be attributed to differences in the extent of post-translational N-linked glycosylation of the enzyme.

Animals↗

Impaired overload-induced muscle growth is associated with diminished translational signalling in aged rat fast-twitch skeletal muscle.

Impaired overload-induced protein synthesis and growth in aged fast-twitch skeletal muscle may result from diminished responsiveness of signalling intermediates controlling protein translation. Yet, potential age-related signalling decrements have never been examined in direct parallel with impaired overload-induced muscle growth in any model. To this end, we used Western blotting to examine the contents and phosphorylation states of mammalian target of rapamycin (mTOR) and its downstream translational signalling intermediates, 70 kDa ribosomal protein S6 kinase (S6k), ribosomal protein S6 (rpS6), eukaryotic elongation factor 2 (eEF2), and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), in conjunction with impaired growth in 1 week overloaded fast-twitch plantaris muscles (via unilateral gastrocnemius ablation) of old (O; 30 months) versus young adult (YA; 8 months) male Fischer344 x Brown Norway rats. The significantly (P <or= 0.05) diminished growth (assessed by total muscle protein content) in overloaded O muscles (5.6 +/- 1.7 versus 19.3 +/- 2.9% in YA) was accompanied by significant impairments in the phosphorylation states of mTOR (Ser2448), S6k (impaired at the mTOR-specific Thr389 residue but not at Thr421/Ser424), rpS6 (Ser235/236) and 4E-BP1 (gel shift), as well as deficits in total eEF2 accretion. Moreover, in overloaded muscles across both age groups, phospho-S6k at Thr389 (but not at Thr421/Ser424), 4E-BP1 phosphorylation status, and total eEF2 accretion were all positively correlated with percentage muscle hypertrophy, and negatively correlated with the phosphorylation (Thr172) of 5'-AMP-activated protein kinase (AMPK; which inhibits translational signalling and protein synthesis in young muscle at rest). As previously published by ourselves, AMPK was hyperphosphorylated in O versus YA muscles used in the current investigation. The present results provide solid evidence that impaired overload-induced growth in aged fast-twitch muscle may partly result from multiple-level decrements in signalling pathway(s) controlling protein translation, and also provide an initial indication that AMPK hyperactivation with age may potentially lie upstream of these decrements.

Adaptation, Physiological↗

[Ubiquitins, proteasomes, sumoylation and therapeutic application today and in future for cancer and other diseases. I. Ubiquitin-proteasome system and the transcription factor NF-kappaB].

Proteasome is protein complex with proteolytic activity. Proteasomes are in addition to lysosomes the main proteolytic machinery of the eukaryotic cell. Proteins destined for degradation in proteasomes are marked by ubiquitinylation, which consists in attachment of polyubiquitin to relevant protein. The transport of polyubiquitinylated protein follows to proteasome, where protein is cleaved into small peptides. Besides polyubiquitin attachment to protein, monoubiquitinylation of proteins exists and has an important role in DNA repair, transcription of genes, endocytosis and signal transduction. The function of an important transcription factor NF-kappaB is connected with proteasome. NF-kappaB is activated after the proteolysis of its inhibitor IkappaB in proteasome. Ubiqutinylation and degradation of protein in proteasome and the activation of NF-kappaB play significant roles in taking proteins away and in expression of great numbers of genes important for the regulation of the cell cycle and apoptosis of cells. The inhibition of proteasomes has antiproliferative and antiinflammatory effects and opens new therapeutic approaches to a treatment of cancer and some inflammatory diseases. We divided the review into three parts: I. Ubiquitin-proteasome system and the transcription factor NF-kappaB, II. Sumoylation and neddylation as post-translational modification of proteins similar to ubiquitinylation and their significance and lastly III. Using of the knowledge of ubiquitin-proteasome system in cancer and other diseases therapy.

Animals↗

Multiple-target drugs: inhibitors of heat shock protein 90 and of histone deacetylase.

In spite of the improvement of conventional medical therapy for cancer treatment, the impact on cancer related mortality in the last ten years has been modest especially for advanced disease in adults. On the other hand, understanding of molecular events underlining tumor development lead to the definition of new molecular targets for novel anti-tumor therapeutical approaches. On this regard, several biotechnology products selected by academic as well as industrial research are currently in clinical trials. Epigenetics as well as post-translational modifications of proteins are emerging as novel attractive targets for anticancer therapy. In addition, the heterogeneity of tumor cells within a selected neoplastic lesions as well as the redundancy of proliferative and survival pathways present in cancer cells favor the development of single drugs that are able to affect multiple pathways. Inhibitors of heat shock protein 90 and of histone deacetylase are two novel classes of multi-target agents that entered recently in clinical studies. This review will focus on the most important issues in the development of both these classes of agents.

Animals↗

En masse analysis of nascent translation using microarrays.

We report a robust method for studying en masse changes in translation using cDNA arrays. The relative distribution of messenger RNAs (mRNAs) along polysome gradients was monitored by performing cDNA array analysis of each gradient fraction and quantifying the mRNA translational status by regression analysis. Using this strategy to study human carcinoma cells exposed to short-wavelength ultraviolet light (UVC), we identified a subset of 17 translationally induced mRNAs and a subset of 69 translationally repressed mRNAs following UVC irradiation. We describe an effective approach for globally investigating changes in protein biosynthesis.

Cell Line, Tumor↗

Aminoglycoside-induced translational read-through in disease: overcoming nonsense mutations by pharmacogenetic therapy.

A third of inherited diseases result from premature termination codon mutations. Aminoglycosides have emerged as vanguard pharmacogenetic agents in treating human genetic disorders due to their unique ability to suppress gene translation termination induced by nonsense mutations. In preclinical and pilot clinical studies, this therapeutic approach shows promise in phenotype correction by promoting otherwise defective protein synthesis. The challenge ahead is to maximize efficacy while preventing interaction with normal protein production and function.

Aminoglycosides↗

Tackling the phosphoproteome: tools and strategies.

Characterization of post-translational modifications in proteins is one of the major tasks that is to be accomplished in the post-genomic era. Phosphorylation is a key reversible modification that regulates enzymatic activity, subcellular localization, complex formation and degradation of proteins. Rapid advances in mass spectrometry instrumentation coupled to the development of analytical methods over the past several years now allow us to investigate the phosphoproteome on a global scale.

Chromatography, High Pressure Liquid↗

Isoelectric focusing as a tool for the investigation of post-translational processing and chemical modifications of proteins.

It has been demonstrated that good agreement may be observed between computed and experimental isoelectric point (pI) values when proteins of known sequence are focused under denaturing conditions on immobilized pH gradient IPG slabs, at least in the pH range 4-7.5. Hence, discrepancies between expected and found in this experimental set-up may be reliably ascribed to some kind of post-transcriptional processing, or chemical modification, having taken place in the sample. This evaluation is made easier when the comparison is set between the pI of a parent molecule and that (or those) of one to several of its derivatives as resolved in a single experiment (for instance, as a spot row in two-dimensional maps); no previous knowledge is required in these cases about the amino acid composition of the primary structure. The effects on protein surface charge are discussed in this review mainly for two biologically relevant processes, glycosylation and phosphorylation. Then, the pI shifts are analysed for some protein modifications that may occur naturally but can also be artefactually elicited, such as NH2 terminus blocking, deamidation and thiol redox reactions. Finally, carboxymethylation and carbamylation are used to exemplify chemical treatments often applied in connection with electrophoretic techniques and involving charged residues. Procedures to be applied in order to verify whether a given modification has occurred, and often relying on the focusing of a treated specimen, are detailed in each section. Numerical examples on model proteins are also discussed. As an important field of application of the above concepts may be genetic engineering, an exhaustive bibliographic list dealing with pI evaluation and structural assessment on recombinant proteins is included.

Animals↗

Precursor structure of cephalosporin acylase. Insights into autoproteolytic activation in a new N-terminal hydrolase family.

Autocatalytic proteolytic cleavage is a frequently observed post-translational modification in proteins. Cephalosporin acylase (CA) is a recently identified member of the N-terminal hydrolase family that is activated from an inactive precursor by autoproteolytic processing, generating a new N-terminal residue, which is either a Ser or a Thr. The N-terminal Ser or Thr becomes a nucleophilic catalytic center for intramolecular and intermolecular amide cleavages. The gene structure of the open reading frame of CAs generally consists of a signal peptide followed by the alpha-subunit, a spacer sequence, and the beta-subunit, which are all translated into a single polypeptide chain, the CA precursor. The precursor is post-translationally modified into an active heterodimeric enzyme with alpha- and beta-subunits, first by intramolecular cleavage and second by intermolecular cleavage. We solved the first CA precursor structure (code 1KEH) from a class I CA from Pseudomonas diminuta at a 2.5-A resolution that provides insight into the mechanism of intramolecular cleavage. A conserved water molecule, stabilized by four hydrogen bonds in unusual pseudotetrahedral geometry, plays a key role to assist the OG atom of Ser(1beta) to generate a strong nucleophile. In addition, the site of the secondary intermolecular cleavage of CA is proposed to be the carbonyl carbon of Gly(158alpha) (Kim, S., and Kim, Y., (2001) J. Biol. Chem., 276, 48376-48381), which is different from the situation in two other class I CAs.

Binding Sites↗