Biomedical subjects

Ulf Hellman

Publications and source records attributed to Ulf Hellman.

54 records · Page 3Linked to original sources

PubMed2003

The protein kinase 60S is a free catalytic CK2alpha' subunit and forms an inactive complex with superoxide dismutase SOD1.

The 60S ribosomes from Saccharomyces cerevisiae contain a set of acidic P-proteins playing an important role in the ribosome function. Reversible phosphorylation of those proteins is a mechanism regulating translational activity of ribosomes. The key role in regulation of this process is played by specific, second messenger-independent protein kinases. The PK60S kinase was one of the enzymes phosphorylating P-proteins. The enzyme has been purified from yeast and characterised. Pure enzyme has properties similar to those reported for casein kinase type 2. Peptide mass fingerprinting (PMF) has identified the PK60S as a catalytic alpha(') subunit of casein kinase type 2 (CK2alpha(')). Protein kinase activity is inhibited by SOD1 and by highly specific CK2 inhibitor-4,5,6,7-tetrabromo-benzotriazole (TBBt). The possible mechanism of regulation of CK2alpha(') activity in stress conditions, by superoxide dismutase in regulation of 80S-ribosome activity, is discussed.

Casein Kinase II↗

PubMed2003

Differential ubiquitination defines the functional status of the tumor suppressor Smad4.

Smad4 is an essential signal transducer of all transforming growth factor-beta (TGF-beta) superfamily pathways that regulate cell growth and differentiation, and it becomes inactivated in human cancers. Receptor-activated (R-) Smads can be poly-ubiquitinated in the cytoplasm or the nucleus, and this regulates their steady state levels or shutdown of the signaling pathway. Oncogenic mutations in Smad4 and other Smads have been linked to protein destabilization and proteasomal degradation. We analyzed a panel of missense mutants derived from human cancers that map in the N-terminal Mad homology (MH) 1 domain of Smad4 and result in protein instability. We demonstrate that all mutants exhibit enhanced poly-ubiquitination and proteasomal degradation. In contrast, wild type Smad4 is a relatively stable protein that undergoes mono- or oligo-ubiquitination, a modification not linked to protein degradation. Analysis of Smad4 deletion mutants indicated efficient mono- or oligo-ubiquitination of the C-terminal MH2 domain. Mass spectrometric analysis of mono-ubiquitinated Smad4 MH2 domain identified lysine 507 as a major target for ubiquitination. Lysine 507 resides in the conserved L3 loop of Smad4 and participates in R-Smad C-terminal phosphoserine recognition. Mono- or oligo-ubiquitinated Smad4 exhibited enhanced ability to oligomerize with R-Smads, whereas mutagenesis of lysine 507 led to inefficient Smad4/R-Smad hetero-oligomerization and defective transcriptional activity. Finally, overexpression of a mutant ubiquitin that only leads to mono-ubiquitination of Smad4 enhanced Smad transcriptional activity. These data suggest that oligo-ubiquitination positively regulates Smad4 function, whereas poly-ubiquitination primarily occurs in unstable cancer mutants and leads to protein degradation.

Amino Acid Sequence↗

PubMed2003

A novel trypsin inhibitor from Peltophorum dubium seeds, with lectin-like properties, triggers rat lymphoma cell apoptosis.

A trypsin inhibitor (PDTI) was isolated from Peltophorum dubium seeds by affinity chromatography on a thyroglobulin-agarose or a trypsin-agarose column. In both cases, SDS-PAGE showed two bands of M(r) 20,000 and 22,000, which could not be resolved. Their amino-terminal sequences were identical and similar to that of Kunitz-type soybean trypsin inhibitor (SBTI). Mass spectrometry analysis of tryptic digests of both bands showed 16 coincident peaks, suggesting that they are closely related proteins. The K(i)s for trypsin and chymotrypsin inhibitory activity of PDTI were 1.6 x 10(-7) and 1.3 x 10(-5)M, respectively. Lectin-like activity of PDTI and SBTI, detected by hemagglutination of rabbit erythrocytes, was inhibited by sialic acid-containing compounds. PDTI and SBTI caused apoptosis of Nb2 rat lymphoma cells, demonstrated by decrease of viability, DNA hypodiploidy, DNA fragmentation, and caspase-3-like activity. They had no effect on normal mouse splenocytes or lymphocytes, whereas they caused apoptosis of concanavalin A-stimulated mouse lymphocytes.

Amino Acid Sequence↗

PubMed2003

Echinococcus granulosus antigen 5 is closely related to proteases of the trypsin family.

Antigen 5 (Ag5) is a dominant secreted component of the larval stage of Echinococcus granulosus, and is highly immunogenic in human infections. Although the diagnostic value of Ag5 has been thoroughly evaluated, there has been little progress in its molecular characterization and the understanding of its biological role. In the present study, the Ag5 gene was cloned by reverse transcription-PCR on the basis of the amino acid sequences of tryptic fragments. The nucleotide sequence indicates that Ag5 is synthesized as a single polypeptide chain that is afterwards processed into single disulphide-bridged 22 and 38 kDa subunits. Whereas the 22 kDa component contains a highly conserved glycosaminoglycan-binding motif that may help to confine Ag5 in the host tissue surrounding the parasite, the 38 kDa subunit is closely related to serine proteases of the trypsin family. The sequences in the vicinity of the active-site histidine, aspartic acid and serine residues, and critical cysteine residues involved in disulphide formation, are well conserved, but the catalytic serine residue is replaced by threonine. Since there are no significant chemical differences between the O gamma atoms of these residues, we performed a series of enzymic assays to find out whether Ag5 is a catalytic molecule. Neither proteolytic activity nor binding to protease inhibitors could be detected using the native purified antigen. Thus it may be possible that Ag5 possesses a highly specific physiological substrate or, more likely, that trypsin-like folding has been recruited to fulfil novel functions.

Amino Acid Sequence↗

PubMed2003

Characterization of a lysosomal serine carboxypeptidase from Trypanosoma cruzi.

Trypanosoma cruzi, the flagellate protozoan which is the causative agent of the American trypanosomiasis, Chagas disease has carboxypeptidase activity. The enzyme has been purified to protein homogeneity, and shown to be a lysosomal monomeric glycoprotein with a molecular mass of about 54kDa. The enzyme has an optimum acidic pH (4.5 with furyl acryloyl-Phe-Phe as substrate), is highly specific for hydrophobic C-terminal amino acid residues, and is strongly inhibited by 3,4-dichloroisocoumarin (IC(50) value 0.3 microM). The enzyme is encoded by a number of genes arrayed in head-to-tail tandems; one of these genes has been cloned and sequenced. Sequence comparisons indicate that the enzyme belongs to the C group of serine carboxypeptidases, within the S10 serine peptidase family, and shows the higher similarity to plant and yeast enzymes. The residues involved in catalysis and most of those involved in substrate binding are conserved in the T. cruzi enzyme as well as 8 out of 10 Cys residues known to be involved in disulfide bridges in the yeast enzyme. This is the first report of an S10 family enzyme in trypanosomatids. The presence of serine carboxypeptidases is not restricted to T. cruzi, being possibly a general character of trypanosomatids.

Amino Acid Sequence↗

PubMed2002

Structural and immunological aspects of Polybia scutellaris Antigen 5.

Vespid venoms contain Antigen 5, an important allergen whose primary structure and immunological behavior have been extensively studied from venoms of vespids of the Northern Hemisphere. We report herein structural and immunological aspects of Antigen 5 from Polybia scutellaris subspecies rioplatensis (vulgar name: camoati) found in South America. Mast cell degranulation, histamine release, and IgE induction experiments performed in mice allow us to suggest that P. scutellaris Antigen 5 is a variant with reduced IgE response and anaphylactic activity. Sequence data indicate that the protein has a 72.5-90.3% similarity to that of members of the vespid Antigen 5 family with an already known primary structure. Moreover, results suggest that the protein-a new member of an extracellular protein superfamily-could be a good candidate for immunotherapy related to vespid allergy.

Allergens↗

PubMed2002

The final N-terminal trimming of a subaminoterminal proline-containing HLA class I-restricted antigenic peptide in the cytosol is mediated by two peptidases.

The proteasome produces MHC class I-restricted antigenic peptides carrying N-terminal extensions, which are trimmed by other peptidases in the cytosol or within the endoplasmic reticulum. In this study, we show that the N-terminal editing of an antigenic peptide with a predicted low TAP affinity can occur in the cytosol. Using proteomics, we identified two cytosolic peptidases, tripeptidyl peptidase II and puromycin-sensitive aminopeptidase, that trimmed the N-terminal extensions of the precursors produced by the proteasome, and led to a transient enrichment of the final antigenic peptide. These peptidases acted either sequentially or redundantly, depending on the extension remaining at the N terminus of the peptides released from the proteasome. Inhibition of these peptidases abolished the CTL-mediated recognition of Ag-expressing cells. Although we observed some proteolytic activity in fractions enriched in endoplasmic reticulum, it could not compensate for the loss of tripeptidyl peptidase II/puromycin-sensitive aminopeptidase activities.

Acetylcysteine↗

PubMed2002

Inhibition of yeast ribosomal stalk phosphorylation by Cu-Zn superoxide dismutase.

Reversible phosphorylation of acidic ribosomal proteins of Saccharomyces cerevisiae is an important mechanism, regulating the number of active ribosomes. The key role in regulation of this process is played by specific, second messenger-independent protein kinases. A new protein-inhibitor regulating activity of PK60S kinase has been purified from yeast extracts and characterised. Peptide mass fingerprinting (PMF) and amino-acid sequence analysis by Post Source Decay (PSD) have identified the inhibitor as a Cu-Zn superoxide dismutase (SOD). Inhibition by SOD is competitive with respect to protein substrates-P proteins and 80S ribosome-with K(i) values of 3.7 microM for P2A protein and 0.6 microM for 80S ribosomes. A close correlation was found between the state of phosphorylation of P proteins in diauxic shift and logarithmic growth yeast cells and activity of SOD. The possible mechanism of regulation of PK60S activity, and participation of SOD protein in regulation of 80S-ribosome activity in stress conditions, is discussed.

Enzyme Inhibitors↗

PubMed2002

Functional proteomics of transforming growth factor-beta1-stimulated Mv1Lu epithelial cells: Rad51 as a target of TGFbeta1-dependent regulation of DNA repair.

Transforming growth factor-beta (TGFbeta) conveys regulatory signals through multiple intracellular pathways, subsequently affecting various cellular functions. To identify new targets for TGFbeta, we studied the changes in the proteome of Mv1Lu lung epithelial cells in response to TGFbeta1 treatment. Thirty-eight non-abundant protein spots, affected by TGFbeta1, were selected, and proteins were identified by peptide mass-fingerprinting (PMF). Among them, proteins involved in regulation of immune response, apoptosis, regulation of TGFbeta signalling, metabolism and DNA repair were identified. Twenty-eight of the 38 proteins are new targets for TGFbeta1, thus suggesting novel ways of integration of TGFbeta signalling in intracellular regulatory processes. We show that TGFbeta1-dependent decrease in expression of one of the new targets, Rad51, correlates with a decrease in DNA repair efficiency. This was evaluated by formation of nuclear Rad51-containing DNA repair complexes in response to DNA damage, by single cell gel electrophoresis and by cell survival assay. The TGFbeta1-dependent inhibition of DNA repair was reversed by ectopic overexpression of Rad51. Therefore, TGFbeta can promote DNA instability through down-regulation of Rad51 and inhibition of DNA repair.

Animals↗

PubMed2002

Easy amino acid sequencing of sulfonated peptides using post-source decay on a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer equipped with a variable voltage reflector.

Tryptic peptides were labeled with sulfonic acid groups at the N-termini using an improved chemistry. The derivatization was performed in common aqueous buffers on peptides adsorbed onto a ZipTip trade mark C(18), thus allowing simultaneous desalting/concentration of the sample. When only Arg-terminating peptides were considered, the procedure from adsorption onto the ZipTip until analysis by MALDI-PSD took about 10 min and several samples could be worked on in parallel. The resulting improved post-source decay (PSD) fragmentation produced spectra containing only y-ions. PSD amino acid sequencing of underivatized and derivatized synthetic peptides was compared. From the sequence information obtained from derivatized peptides isolated by ion selection from tryptic in-gel digests, a protein was correctly identified which was difficult to analyze from an unclear peptide mass fingerprint analysis. The method was also applied to the identification and localization of phosphorylated Ser and Tyr residues in native and synthetic peptides.

Guanidines↗

PubMed2002

Control of Smad7 stability by competition between acetylation and ubiquitination.

Smad proteins regulate gene expression in response to TGFbeta signaling. Here we present evidence that Smad7 interacts with the transcriptional coactivator p300, resulting in acetylation of Smad7 on two lysine residues in its N terminus. Acetylation or mutation of these lysine residues stabilizes Smad7 and protects it from TGFbeta-induced degradation. Furthermore, we demonstrate that the acetylated residues in Smad7 also are targeted by ubiquitination and that acetylation of these lysine residues prevents subsequent ubiquitination. Specifically, acetylation of Smad7 protects it against ubiquitination and degradation mediated by the ubiquitin ligase Smurf1. Thus, our data suggest that competition between ubiquitination and acetylation of overlapping lysine residues constitutes a novel mechanism to regulate protein stability.

Acetylation↗

PubMed2002

Cloning of the manganese lipoxygenase gene reveals homology with the lipoxygenase gene family.

Manganese lipoxygenase was isolated to homogeneity from the take-all fungus, Gaeumannomyces graminis. The C-terminal amino acids and several internal peptides were sequenced, and the information was used to obtain a cDNA probe by RT/PCR. Screening of a genomic library of G. graminis yielded a full-length clone of the Mn-Lipoxygenase gene. cDNA analysis showed that the gene spanned 2.6 kb and contained one intron (133 bp). Northern blot analyses indicated two transcripts (2.7 and 3.1 kb). The deduced amino-acid sequence of the Mn-Lipoxygenase precursor (618 amino acids, 67.7 kDa) could be aligned with mammalian and plant lipoxygenases with 23-28% identity over 350-400 amino-acid residues of the catalytic domains. Lipoxygenases have one water molecule and five amino acids as Fe ligands. These are two histidine residues in the highly conserved 30 amino-acid sequence WLLAK-X15-H-X4-H-X3-E of alpha helix 9, one histidine and usually an asparaine residue in the sequence H-X3-N-X-G of alpha helix 18, and the carboxyl oxygen of the C-terminal isoleucine (or valine) residue. The homologous sequence of alpha helix 9 of Mn-Lipoxygenase [WLLAK-X14-H(294)-X3-H(297)-X3-E] contained two single-amino-acid gaps, but otherwise His294 and His297 aligned with the two His residues, which coordinate iron. Mn-Lipoxygenase [H(478)-X3-N(482)-X-G] could be aligned with the two metal ligands of alpha helix 18, and the C-terminal residue was Val618. We conclude that Mn-Lipoxygenase belongs to the lipoxygenase gene family and that its unique biochemical properties might be related to structural differences in the metal centre and alpha helix 9 of lipoxygenases rather than to the metal ligands.

Amino Acid Sequence↗

PubMed2002

Mitochondrial ATP synthase--a possible target protein in the regulation of energy metabolism in vitro and in vivo.

The increasing prevalence of obesity in the Western world has stimulated an intense search for mechanisms regulating food intake and energy balance. A number of appetite-regulating peptides have been identified, their receptors cloned and the intracellular events characterized. One possible energy-dissipating mechanism is the mitochondrial uncoupling of ATP-synthesis from respiratory chain oxidation through uncoupling proteins, whereby energy derived from food could be dissipated as heat, instead of stored as ATP. The exact role of the uncoupling proteins in energy balance is, however, uncertain. We show here that mitochondrial F1F0-ATP synthase itself is a target protein for an anorectic peptide, enterostatin, demonstrated both after affinity purification of rat brain membranes and through a direct physical interaction between enterostatin and purified F1-ATP synthase. In insulinoma cells (INS-1) enterostatin was found to target F1F0-ATP synthase, causing an inhibition of ATP production, an increased thermogenesis and increased oxygen consumption. The experiments suggest a role of mitochondrial F1F0-ATP synthase in the suppressed insulin secretion induced by enterostatin. It could be speculated that this targeting mechanism is involved in the decreased energy efficiency following enterostatin treatment in rat.

Animals↗

PubMed2002

Evidence for a posttranscriptional role of a TFIIICalpha-like protein in Chironomus tentans.

We have cloned and sequenced a cDNA that encodes for a nuclear protein of 238 kDa in the dipteran Chironomus tentans. This protein, that we call p2D10, is structurally similar to the alpha subunit of the general transcription factor TFIIIC. Using immunoelectron microscopy we have shown that a fraction of p2D10 is located at sites of transcription, which is consistent with a possible role of this protein in transcription initiation. We have also found that a large fraction of p2D10 is located in the nucleoplasm and in the nuclear pore complexes. Using gel filtration chromatography and coimmunoprecipitation methods, we have identified and characterized two p2D10-containing complexes that differ in molecular mass and composition. The heavy p2D10-containing complex contains at least one other component of the TFIIIC complex, TFIIIC-epsilon. Based on its molecular mass and composition, the heavy p2D10-containing complex may be the Pol III holoenzyme. The light p2D10-containing complex contains RNA together with at least two proteins that are thought to be involved in mRNA trafficking, RAE1 and hrp65. The observations reported here suggest that this new TFIIIC-alpha-like protein is involved in posttranscriptional steps of premRNA metabolism in Chironomus tentans.

Amino Acid Motifs↗

PubMed2002

A novel conserved RNA-binding domain protein, RBD-1, is essential for ribosome biogenesis.

Synthesis of the ribosomal subunits from pre-rRNA requires a large number of trans-acting proteins and small nucleolar ribonucleoprotein particles to execute base modifications, RNA cleavages, and structural rearrangements. We have characterized a novel protein, RNA-binding domain-1 (RBD-1), that is involved in ribosome biogenesis. This protein contains six consensus RNA-binding domains and is conserved as to sequence, domain organization, and cellular location from yeast to human. RBD-1 is essential in Caenorhabditis elegans. In the dipteran Chironomus tentans, RBD-1 (Ct-RBD-1) binds pre-rRNA in vitro and anti-Ct-RBD-1 antibodies repress pre-rRNA processing in vivo. Ct-RBD-1 is mainly located in the nucleolus in an RNA polymerase I transcription-dependent manner, but it is also present in discrete foci in the interchromatin and in the cytoplasm. In cytoplasmic extracts, 20-30% of Ct-RBD-1 is associated with ribosomes and, preferentially, with the 40S ribosomal subunit. Our data suggest that RBD-1 plays a role in structurally coordinating pre-rRNA during ribosome biogenesis and that this function is conserved in all eukaryotes.

Animals↗

PubMedDate not supplied

Identification of the chicken MARCKS phosphorylation site specific for differentiating neurons as Ser 25 using a monoclonal antibody and mass spectrometry.

MARCKS is an actin-modulating protein that can be phosphorylated in multiple sites by PKC and proline-directed kinases. We have previously described a phosphorylated form of this protein specific for differentiating chick neurons, detected with mAb 3C3. Here, we show that this antibody binds to MARCKS only when it is phosphorylated at Ser 25. These and previous data provide hints for a possible answer to the question of why this ubiquitous protein seems to be essential only for neural development.

Animals↗

PubMedDate not supplied

ITREOH building of regional capacity to monitor recreational water: development of a non-commercial microcystin ELISA and its impact on public health policy.

In 2001, a University of California, Davis-University of the Republic, Montevideo, partnership created a Fogarty ITREOH program to exploit the potential of ELISA to provide a low-cost environmental analysis attractive to economically distressed countries of temperate South America. This paper describes the development and validation of an ELISA method for the determination of Cyanobacteria microcystin toxins in algal blooms, which release hepatotoxic metabolites that can reach toxic levels in rivers, lakes, or coastal estuaries used for recreation or water supplies. The assay made possible the first systematic monitoring of water from the Rio de la Plata at Montevideo over two summers. The project has been integrated into a bi-national effort to monitor the Rio de la Plata.

Bacterial Toxins↗

PubMedDate not supplied

Subcellular fractionation and electrophoretic analysis of proteins from the regenerating nerve in rabbits following treatment with triiodothyronine (T3).

BACKGROUND: Triidothyronine(T3) plays a critical role in the physiological function of virtually all tissues. In the present study the effect of T3 on nerve regeneration was studied. MATERIALS AND METHODS: The right sciatic nerve of 20 male rabbits was axonotomised. Ten of the animals were used as controls while the others received T3. Seven days later, all were sacrificed. Both sciatic nerves were excised and each axonotomised nerve was divided into three equal segments: I (proximal to the lesion), II (containing the lesion), III (distal to the lesion). Corresponding segments from the contralateral nerves were used as controls. After subcellular fractionation, proteins were analyzed by polyacrylamide gel electrophoresis and identified by proteomic analysis. RESULTS: T3 administration increased the levels of tubulins alpha 6 and beta 3, beta-actin, myelin protein zero, myelin basic protein, myelin protein-2 and histones H3 and H2A in segments I and III of the injured nerves. Increases in the levels of beta-actin and Y box-binding protein correlated to the event of injury rather than T3 administration. CONCLUSION: Our findings indicate that T3 treatment positively affects peripheral nerve regeneration.

Animals↗