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  • 1. Carlén, Lina M
    et al.
    Sánchez, Fabio
    Bergman, Ann-Charlotte
    Becker, Susanne
    Hirschberg, Daniel
    Franzén, Bo
    Coffey, Jonathan
    Jörnvall, Hans
    Auer, Gert
    Alaiya, Ayodele A
    Ståhle, Mona
    Proteome analysis of skin distinguishes acute guttate from chronic plaque psoriasis.2005In: Journal of Investigative Dermatology, ISSN 0022-202X, E-ISSN 1523-1747, Vol. 124, no 1Article in journal (Refereed)
    Abstract [en]

    Psoriasis is a disease with considerable heterogeneity in clinical presentation. This is the first study using two-dimensional gel electrophoresis to compare global protein expression patterns in lesional and non-lesional skin from subjects with acute guttate psoriasis associated with streptococcal throat infection and chronic plaque psoriasis. Samples from experimentally induced contact eczema and normal skin from healthy controls were also included. Proteins with statistically significant differences in expression were used in hierarchical cluster analyses resulting in separation of the different samples into groups. Chronic plaque and guttate psoriasis samples were distinctly separated, indicating that they represent discrete phenotypes at the protein expression level. Interestingly, there was a trend in which guttate psoriasis lesions clustered closer to eczema than to chronic plaque psoriasis lesions, indicating that the duration of the inflammatory reaction may affect clustering. Several of the differentially expressed proteins were identified by mass spectrometry.

  • 2. Christiansen, Maja
    et al.
    Jørgensen, Charlotte S
    Laursen, Inga
    Hirschberg, Daniel
    Højrup, Peter
    Houen, Gunnar
    Protein chemical characterization of Gc globulin (vitamin D-binding protein) isoforms; Gc-1f, Gc-1s and Gc-2.2007In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1774, no 4Article in journal (Refereed)
    Abstract [en]

    Gc globulin, also called vitamin D-binding protein, is a plasma protein involved in the extracellular actin-scavenger system, vitamin D transport and possibly also other biological activities. Low levels of Gc globulin have been found to correlate with multiple organ failure and non-survival of patients with fulminant hepatic failure and trauma. Here, we characterize the dominant isoforms of plasma-derived Gc globulin from Cohn fraction IV paste with respect to amino acid sequence and posttranslational modifications. Gc globulin was purified in large scale and the isoforms separated by ion exchange chromatography. The separated isoforms and several commercial preparations of individual isoforms were characterized by mass spectrometry. This revealed that the major isoforms were non-glycosylated. Compared to the Gc-1f isoform the other dominating isoforms represented an Asp/Glu substitution (Gc-1s) and a Thr/Lys substitution (Gc-2) in agreement with DNA sequencing studies. The commercial preparations were found to represent mainly one or two isoforms. An O-linked glycan with a mass of 656 Da and terminating with a sialic acid residue was detected on a minor proportion of Gc globulin molecules.

  • 3. Filling, Charlotta
    et al.
    Keller, Brigitte
    Hirschberg, Daniel
    Marschall, Hanns-Ulrich
    Jörnvall, Hans
    Bennett, Michael J
    Oppermann, Udo
    Role of short-chain hydroxyacyl CoA dehydrogenases in SCHAD deficiency.2008In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 368, no 1Article in journal (Refereed)
    Abstract [en]

    Short-chain hydroxyacyl CoA dehydrogenase deficiency is an ill-defined, severe pediatric disorder of mitochondrial fatty acid beta-oxidation of short-chain hydroxyacyl CoAs. To understand the relative contributions of the two known short-chain hydroxyacyl CoA dehydrogenases (HADH) tissue biopsies of six distinct family individuals were analyzed and kinetic parameters were compared. Steady-state kinetic constants for HADH 1 and HADH 2 suggest that type 1 is the major enzyme involved in mitochondrial beta-oxidation of short-chain hydroxyacyl-CoAs. Two patients are heterozygous carriers of a HADH 1 polymorphism, whereas no mutation is detected in the HADH 2 gene of all patients. The data suggest that protein interactions rather than HADH mutations are responsible for the disease phenotype. Pull-down experiments of recombinant HADH 1 and 2 with human mitochondrial extracts reveal two proteins interacting with HADH 1, one of which was identified as glutamate dehydrogenase. This association provides a possible link between fatty acid metabolism and the hyperinsulinism/hyperammonia syndrome.

  • 4. Groenning, Minna
    et al.
    Campos, Raul I
    Hirschberg, Daniel
    IFM – Department of Chemistry, Linköping University, Linköping, Sweden.
    Hammarström, Per
    Vestergaard, Bente
    Considerably Unfolded Transthyretin Monomers Preceed and Exchange with Dynamically Structured Amyloid Protofibrils2015In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 5, article id 11443Article in journal (Refereed)
    Abstract [en]

    Despite numerous studies, a detailed description of the transthyretin (TTR) self-assembly mechanism and fibril structure in TTR amyloidoses remains unresolved. Here, using a combination of primarily small -angle X-ray scattering (SAXS) and hydrogen exchange mass spectrometry (HXMS) analysis, we describe an unexpectedly dynamic TTR protofibril structure which exchanges protomers with highly unfolded monomers in solution. The protofibrils only grow to an approximate final size of 2,900 kDa and a length of 70 nm and a comparative HXMS analysis of native and aggregated samples revealed a much higher average solvent exposure of TTR upon fibrillation. With SAXS, we reveal the continuous presence of a considerably unfolded TTR monomer throughout the fibrillation process, and show that a considerable fraction of the fibrillating protein remains in solution even at a late maturation state. Together, these data reveal that the fibrillar state interchanges with the solution state. Accordingly, we suggest that TTR fibrillation proceeds via addition of considerably unfolded monomers, and the continuous presence of amyloidogenic structures near the protofibril surface offers a plausible explanation for secondary nucleation. We argue that the presence of such dynamic structural equilibria must impact future therapeutic development strategies.

  • 5. Gustafsson, Magnus
    et al.
    Hirschberg, Daniel
    Palmberg, Carina
    Jörnvall, Hans
    Bergman, Tomas
    Integrated sample preparation and MALDI mass spectrometry on a microfluidic compact disk.2004In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 76, no 2Article in journal (Refereed)
    Abstract [en]

    High-throughput microfluidic processing of protein digests integrated with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry on a compact disk (CD) is described. Centrifugal force moves liquid through multiple microstructures, each containing a 10-nL reversed-phase chromatography column. The CD enables parallel preparation of 96 samples with volumes ranging from one to several microliters. The peptides in the digests are concentrated, desalted, and subsequently eluted from the columns directly into MALDI target areas (200 x 400 microm) on the CD using a solvent containing the MALDI matrix. After crystallization, the CD is inserted into the MALDI instrument for peptide mass fingerprinting and database identification at a routine sensitivity down to the 200-amol level. Detection of proteolytic peptides down to the 50-amol level is demonstrated. The success rate of the CD technology in protein identification is about twice that of the C(18) ZipTips and standard MALDI steel targets. The CDs are operated using robotics to transfer samples and reagents from microcontainers to the processing inlets on the disposable CD and spinning to control the movement of liquid through the microstructures.

  • 6. Hattula, Katarina
    et al.
    Hirschberg, Daniel
    Kalkkinen, Nisse
    Butcher, Sarah J
    Ora, Ari
    Association between the intrinsically disordered protein PEX19 and PEX3.2014In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 7Article in journal (Refereed)
    Abstract [en]

    In peroxisomes, peroxins (PEXs) 3 and 19 are the principal protein components of the machinery required for early peroxisomal biogenesis. For further insight into the interaction of PEX3 and PEX19, we used hydrogen exchange mass spectrometry to monitor conformational changes during complex formation between PEX3 and PEX19 in vitro. Our data showed that PEX19 remained highly flexible during interaction with PEX3. However, we could detect three changes, one each in the N-and C-terminus along with a small stretch in the middle of PEX19 (F64-L74) which became shielded from hydrogen exchange when interacting with PEX3. PEX3 became more protected from hydrogen exchange in the binding groove for PEX19 with only small changes elsewhere. Most likely the N-terminus of PEX19 initiates the binding to PEX3, and then subtle conformational changes in PEX3 affect the surface of the PEX3 molecule. PEX19 in turn, is stabilized by folding of a short helix and its C-terminal folding core permitting PEX19 to bind to PEX3 with higher affinity than just the N-terminal interaction allows. Thus within the cell, PEX3 is stabilized by PEX19 preventing PEX3 aggregation.

  • 7. Hauge, Camilla
    et al.
    Antal, Torben L
    Hirschberg, Daniel
    Doehn, Ulrik
    Thorup, Katrine
    Idrissova, Leila
    Hansen, Klaus
    Jensen, Ole N
    Jørgensen, Thomas J
    Biondi, Ricardo M
    Frödin, Morten
    Mechanism for activation of the growth factor-activated AGC kinases by turn motif phosphorylation.2007In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 26, no 9Article in journal (Refereed)
    Abstract [en]

    The growth factor/insulin-stimulated AGC kinases share an activation mechanism based on three phosphorylation sites. Of these, only the role of the activation loop phosphate in the kinase domain and the hydrophobic motif (HM) phosphate in a C-terminal tail region are well characterized. We investigated the role of the third, so-called turn motif phosphate, also located in the tail, in the AGC kinases PKB, S6K, RSK, MSK, PRK and PKC. We report cooperative action of the HM phosphate and the turn motif phosphate, because it binds a phosphoSer/Thr-binding site above the glycine-rich loop within the kinase domain, promoting zipper-like association of the tail with the kinase domain, serving to stabilize the HM in its kinase-activating binding site. We present a molecular model for allosteric activation of AGC kinases by the turn motif phosphate via HM-mediated stabilization of the alphaC helix. In S6K and MSK, the turn motif phosphate thereby also protects the HM from dephosphorylation. Our results suggest that the mechanism described is a key feature in activation of upto 26 human AGC kinases.

  • 8. Hindie, Valerie
    et al.
    Stroba, Adriana
    Zhang, Hua
    Lopez-Garcia, Laura A
    Idrissova, Leila
    Zeuzem, Stefan
    Hirschberg, Daniel
    Schaeffer, Francis
    Jørgensen, Thomas J D
    Engel, Matthias
    Alzari, Pedro M
    Biondi, Ricardo M
    Structure and allosteric effects of low-molecular-weight activators on the protein kinase PDK1.2009In: Nature Chemical Biology, ISSN 1552-4450, E-ISSN 1552-4469, Vol. 5, no 10Article in journal (Refereed)
    Abstract [en]

    Protein phosphorylation transduces a large set of intracellular signals. One mechanism by which phosphorylation mediates signal transduction is by prompting conformational changes in the target protein or interacting proteins. Previous work described an allosteric site mediating phosphorylation-dependent activation of AGC kinases. The AGC kinase PDK1 is activated by the docking of a phosphorylated motif from substrates. Here we present the crystallography of PDK1 bound to a rationally developed low-molecular-weight activator and describe the conformational changes induced by small compounds in the crystal and in solution using a fluorescence-based assay and deuterium exchange experiments. Our results indicate that the binding of the compound produces local changes at the target site, the PIF binding pocket, and also allosteric changes at the ATP binding site and the activation loop. Altogether, we present molecular details of the allosteric changes induced by small compounds that trigger the activation of PDK1 through mimicry of phosphorylation-dependent conformational changes.

  • 9. Hirschberg, Daniel
    et al.
    Cederlund, E
    Crosas, B
    Jonsson, A
    Tryggvason, S
    Farrés, J
    Parés, X
    Bergman, T
    Jörnvall, H
    N-terminal acetylation in a third protein family of vertebrate alcohol dehydrogenase/retinal reductase found through a 'proteomics' approach in enzyme characterization.2001In: Cellular and Molecular Life Sciences (CMLS), ISSN 1420-682X, E-ISSN 1420-9071, Vol. 58, no 9Article in journal (Refereed)
    Abstract [en]

    A recent finding of a novel class of retinol-active alcohol dehydrogenase (ADH) in frog prompted analysis of this activity in other vertebrate forms. Surprisingly, yet another and still more unrelated ADH was identified in chicken tissues. It was found to be a member of the aldo-keto reductase (AKR) enzyme family, not previously known as an ADH in vertebrates. Its terminal blocking group and the N-terminal segment, not assigned by protein and cDNA structure analysis, were determined by electrospray tandem mass spectrometry after protein isolation by two-dimensional gel electrophoresis. The N terminus is Acetyl-Ala- and the N-terminal segment contains two consecutive Asn residues. The results establish the new ADH enzyme of the AKR family and show the usefulness of combined gel separation and mass spectrometry in enzyme-characterization.

  • 10. Hirschberg, Daniel
    et al.
    Jägerbrink, Theres
    Samskog, Jenny
    Gustafsson, Magnus
    Ståhlberg, Marie
    Alvelius, Gunvor
    Husman, Bolette
    Carlquist, Mats
    Jörnvall, Hans
    Bergman, Tomas
    Detection of phosphorylated peptides in proteomic analyses using microfluidic compact disk technology.2004In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 76, no 19Article in journal (Refereed)
    Abstract [en]

    A compact disk (CD)-based microfluidic method for selective detection of phosphopeptides by mass spectrometry is described. It combines immobilized metal affinity chromatography (IMAC) and enzymatic dephosphorylation. Phosphoproteins are digested with trypsin and processed on the CD using nanoliter scale IMAC with and without subsequent in situ alkaline phosphatase treatment. This is followed by on-CD matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Dephosphorylation of the IMAC-enriched peptides allows selective phosphopeptide detection based on the differential mass maps generated (mass shifts of 80 Da or multiples of 80 Da). The CD contains 96 microstructures, each with a 16 nL IMAC microfluidic column. Movement of liquid is controlled by differential spinning of the disk. Up to 48 samples are distributed onto the CD in two equal sets. One set is for phosphopeptide enrichment only, the other for identical phosphopeptide enrichment but combined with in situ dephosphorylation. Peptides are eluted from the columns directly into MALDI target areas, still on the CD, using a solvent containing the MALDI matrix. After crystallization, the CD is inserted into a MALDI mass spectrometer for analysis down to the femtomole level. The average success rate in phosphopeptide detection is over 90%. Applied to noncharacterized samples, the method identified two novel phosphorylation sites, Thr 735 and Ser 737, in the ligand-binding domain of the human mineralocorticoid receptor.

  • 11. Hirschberg, Daniel
    et al.
    Rådmark, Olof
    Jörnvall, Hans
    Bergman, Tomas
    Thr94 in bovine myelin basic protein is a second phosphorylation site for 42-kDa mitogen-activated protein kinase (ERK2).2003In: Journal of Protein Chemistry, ISSN 0277-8033, E-ISSN 1573-4943, Vol. 22, no 2Article in journal (Refereed)
    Abstract [en]

    Treatment of bovine brain myelin basic protein with 42-kDa mitogen-activated protein kinase [p42 MAPK or extracellular signal-regulated kinase 2 (ERK2)] in the presence of ATP and Mg2+ results in phosphorylation of Thr94 and Thr97. Thr94 is not previously known to be an ERK2 phosphorylation site. Both residues are phosphorylated to about the same extent and are in the highly conserved segment Asn91-Ile-Val-Thr94-Pro-Arg-Thr97-Pro-Pro-Pro-Ser101 MALDI mass spectrometry before and after ERK2 treatment revealed the addition of two phosphate groups to the protein. Tryptic cleavage resulted in a single fragment (positions 91-104) carrying the observed mass increase. Tandem mass spectrometry applied to the tryptic peptide showed that both Thr94 and Thr97 are acceptors of phosphate. A singly phosphorylated species could not be detected. Identification of the ERK2 phosphorylation site Thr94 in bovine myelin basic protein reveals a nontraditional phosphate acceptor position, preceded by three noncharged residues (Asn-Ile-Val). Proline at position -2 or -3 from the phosphorylation site, typical for the recognition sequence of proline-directed kinases, is missing. The results provide information for delineation of a further substrate consensus motif for ERK2 phosphorylation.

  • 12. Hirschberg, Daniel
    et al.
    Tryggvason, Sam
    Gustafsson, Magnus
    Bergman, Tomas
    Swedenborg, Jesper
    Hedin, Ulf
    Jörnvall, Hans
    Identification of endothelial proteins by MALDI-MS using a compact disc microfluidic system.2004In: The Protein Journal, ISSN 1572-3887, E-ISSN 1875-8355, Vol. 23, no 4Article in journal (Refereed)
    Abstract [en]

    Vascular endothelial proteins have been analyzed using two-dimensional (2D) gel electrophoresis and subsequent mass spectrometry, with separate methods for the intervening sample preparations. Compact disc (CD) technology was found to be rapid, giving high overall yield both with ordinary Coomassie staining and with Sypro Ruby staining. Combined with automatic in-gel digestion, the CD technology has great capacity for large numbers of protein analysis, although for limited sample numbers, manual methods can give similar sequence coverage. In a test set of 48 samples, 45 proteins were identified using the CD preparation technique, 32 identified with higher sequence coverage using the CD technique, 7 with higher using ZipTips in a robotic workstation, and 5 with higher coverage using dried droplets of unpurified samples. In the process of these methodological comparisons, basic patterns for 116 endothelial proteins were defined, representing 297 separate protein spots on the 2D gels.

  • 13. Kjaergaard, Magnus
    et al.
    Gårdsvoll, Henrik
    Hirschberg, Daniel
    Nielbo, Steen
    Mayasundari, Anand
    Peterson, Cynthia B
    Jansson, Anna
    Jørgensen, Thomas J D
    Poulsen, Flemming M
    Ploug, Michael
    Solution structure of recombinant somatomedin B domain from vitronectin produced in Pichia pastoris.2007In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 16, no 9Article in journal (Refereed)
    Abstract [en]

    The cysteine-rich somatomedin B domain (SMB) of the matrix protein vitronectin is involved in several important biological processes. First, it stabilizes the active conformation of the plasminogen activator inhibitor (PAI-1); second, it provides the recognition motif for cell adhesion via the cognate integrins (alpha(v)beta(3), alpha(v)beta(5), and alpha(IIb)beta(3)); and third, it binds the complex between urokinase-type plasminogen activator (uPA) and its glycolipid-anchored receptor (uPAR). Previous structural studies on SMB have used recombinant protein expressed in Escherichia coli or SMB released from plasma-derived vitronectin by CNBr cleavage. However, different disulfide patterns and three-dimensional structures for SMB were reported. In the present study, we have expressed recombinant human SMB by two different eukaryotic expression systems, Pichia pastoris and Drosophila melanogaster S2-cells, both yielding structurally and functionally homogeneous protein preparations. Importantly, the entire population of our purified, recombinant SMB has a solvent exposure, both as a free domain and in complex with PAI-1, which is indistinguishable from that of plasma-derived SMB as assessed by amide hydrogen ((1)H/(2)H) exchange. This solvent exposure was only reproduced by one of three synthetic SMB products with predefined disulfide connectivities corresponding to those published previously. Furthermore, this connectivity was also the only one to yield a folded and functional domain. The NMR structure was determined for free SMB produced by Pichia and is largely consistent with that solved by X-ray crystallography for SMB in complex with PAI-1.

  • 14. Lexander, Helena
    et al.
    Franzén, Bo
    Hirschberg, Daniel
    Becker, Susanne
    Hellström, Magnus
    Bergman, Tomas
    Jörnvall, Hans
    Auer, Gert
    Egevad, Lars
    Differential protein expression in anatomical zones of the prostate.2005In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 5, no 10Article in journal (Refereed)
    Abstract [en]

    The prostate has three anatomical zones: the peripheral (PZ), the transition (TZ), and the central (CZ) zone. It is proposed that the CZ may be of mesodermal origin, whereas the other two are of endodermal origin. Proteome patterns in the zones were characterized to test for differences. Cells were scraped from macroscopically normal areas of PZ, TZ, and CZ in radical prostatectomy specimens. After exclusion of samples with cancer or prostatic intraepithelial neoplasia, 18 cases remained for analysis. Cells were collected in a medium with protease inhibitors, and the protein material was prepared for two-dimensional gel electrophoresis. The proteins in spots that differed quantitatively between regions were identified via mass spectrometric fingerprinting of tryptic fragments and selected tandem mass spectrometry sequence analysis. Ten proteins with significant zonal differential expression were identified, eight with underexpression in the CZ versus the PZ and the TZ (arginase II, ATP synthase, cytokeratin 8, lamin A/C, peroxiredoxin 4, protein disulfide isomerase A3, tropomyosin, and vimentin), and two with overexpression in the CZ (peroxiredoxin 2 and creatine kinase B). The PZ and TZ, although differing in terms of incidence of cancer and hyperplasia, have epithelium with highly similar major protein expression profiles. However, the protein profile of the CZ differs from that of the other regions, suggesting functional differences.

  • 15. Marinova, Zoya
    et al.
    Vukojevic, Vladana
    Surcheva, Slavina
    Yakovleva, Tatiana
    Cebers, Gvido
    Pasikova, Natalia
    Usynin, Ivan
    Hugonin, Loïc
    Fang, Weijie
    Hallberg, Mathias
    Hirschberg, Daniel
    Bergman, Tomas
    Langel, Ulo
    Hauser, Kurt F
    Pramanik, Aladdin
    Aldrich, Jane V
    Gräslund, Astrid
    Terenius, Lars
    Bakalkin, Georgy
    Translocation of dynorphin neuropeptides across the plasma membrane. A putative mechanism of signal transmission.2005In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 280, no 28Article in journal (Refereed)
    Abstract [en]

    Several peptides, including penetratin and Tat, are known to translocate across the plasma membrane. Dynorphin opioid peptides are similar to cell-penetrating peptides in a high content of basic and hydrophobic amino acid residues. We demonstrate that dynorphin A and big dynorphin, consisting of dynorphins A and B, can penetrate into neurons and non-neuronal cells using confocal fluorescence microscopy/immunolabeling. The peptide distribution was characterized by cytoplasmic labeling with minimal signal in the cell nucleus and on the plasma membrane. Translocated peptides were associated with the endoplasmic reticulum but not with the Golgi apparatus or clathrin-coated endocytotic vesicles. Rapid entry of dynorphin A into the cytoplasm of live cells was revealed by fluorescence correlation spectroscopy. The translocation potential of dynorphin A was comparable with that of transportan-10, a prototypical cell-penetrating peptide. A central big dynorphin fragment, which retains all basic amino acids, and dynorphin B did not enter the cells. The latter two peptides interacted with negatively charged phospholipid vesicles similarly to big dynorphin and dynorphin A, suggesting that interactions of these peptides with phospholipids in the plasma membrane are not impaired. Translocation was not mediated via opioid receptors. The potential of dynorphins to penetrate into cells correlates with their ability to induce non-opioid effects in animals. Translocation across the plasma membrane may represent a previously unknown mechanism by which dynorphins can signal information to the cell interior.

  • 16. Natan, Eviatar
    et al.
    Hirschberg, Daniel
    Morgner, Nina
    Robinson, Carol V
    Fersht, Alan R
    Ultraslow oligomerization equilibria of p53 and its implications.2009In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 106, no 34Article in journal (Refereed)
    Abstract [en]

    The tumor suppressor p53 is in equilibrium at cellular concentrations between dimers and tetramers. Oncogenic mutant p53 (mut) exerts a dominant-negative effect on co-expression of p53 wild-type (wt) and mut alleles in cancer cells. It is believed that wt and mut form hetero-tetramers of attenuated activity, via their tetramerization domains. Using electrospray mass spectrometry on isotopically labeled samples, we measured directly the composition and rates of formation of p53 complexes in the presence and absence of response element DNA. The dissociation of tetramers was unexpectedly very slow (t(1/2) = 40 min) at 37 degrees C, matched by slow association of dimers, which is approximately four times longer than the half-life of spontaneous denaturation of wt p53. On mixing wt tetramers with the oncogenic contact mutant R273H of low DNA affinity, we observed the same slow formation of only wt(4), wt(2)mut(2), and mut(4), in the ratio 1:2:1, on a cellular time scale. On mixing wt and mut with response element DNAs P21 and BAX, we observed only the complexes wt(4)xDNA, wt(2)mut(2)xDNA, and mut(4)xDNA, with relative dissociation constants 1:4:71 and 1:13:85, respectively, accounting for the dominant-negative effect by weakened affinity. p53 dimers assemble rapidly to tetramers on binding to response element DNA, initiated by the p53 DNA binding domains. The slow oligomerization of free p53, competing with spontaneous denaturation, has implications for the possible regulation of p53 by binding proteins and DNA that affect tetramerization kinetics as well as equilibria.

  • 17. Park, Ah Young
    et al.
    Jergic, Slobodan
    Politis, Argyris
    Ruotolo, Brandon T
    Hirshberg, Daniel
    Jessop, Linda L
    Beck, Jennifer L
    Barsky, Daniel
    O'Donnell, Mike
    Dixon, Nicholas E
    Robinson, Carol V
    A single subunit directs the assembly of the Escherichia coli DNA sliding clamp loader.2010In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 18, no 3Article in journal (Refereed)
    Abstract [en]

    Multi-protein clamp loader complexes are required to load sliding clamps onto DNA. In Escherichia coli the clamp loader contains three DnaX (tau/gamma) proteins, delta, and delta', which together form an asymmetric pentameric ring that also interacts with psichi. Here we used mass spectrometry to examine the assembly and dynamics of the clamp loader complex. We find that gamma exists exclusively as a stable homotetramer, while tau is in a monomer-dimer-trimer-tetramer equilibrium. delta' plays a direct role in the assembly as a tau/gamma oligomer breaker, thereby facilitating incorporation of lower oligomers. With delta', both delta and psichi stabilize the trimeric form of DnaX, thus completing the assembly. When tau and gamma are present simultaneously, mimicking the situation in vivo, subunit exchange between tau and gamma tetramers occurs rapidly to form heterocomplexes but is retarded when deltadelta' is present. The implications for intracellular assembly of the DNA polymerase III holoenzyme are discussed.

  • 18. Rahman-Roblick, Rubaiyat
    et al.
    Roblick, Uwe Johannes
    Hellman, Ulf
    Conrotto, Paolo
    Liu, Tao
    Becker, Susanne
    Hirschberg, Daniel
    Jörnvall, Hans
    Auer, Gert
    Wiman, Klas G
    p53 targets identified by protein expression profiling.2007In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 104, no 13Article in journal (Refereed)
    Abstract [en]

    p53 triggers cell cycle arrest and apoptosis through transcriptional regulation of specific target genes. We have investigated the effect of p53 activation on the proteome using 2D gel electrophoresis analysis of mitomycin C-treated HCT116 colon carcinoma cells carrying wild-type p53. Approximately 5,800 protein spots were separated in overlapping narrow-pH-range gel strips, and 115 protein spots showed significant expression changes upon p53 activation. The identity of 55 protein spots was obtained by mass spectrometry. The majority of the identified proteins have no previous connection to p53. The proteins fall into different functional categories, such as mRNA processing, translation, redox regulation, and apoptosis, consistent with the idea that p53 regulates multiple cellular pathways. p53-dependent regulation of five of the up-regulated proteins, eIF5A, hnRNP C1/C2, hnRNP K, lamin A/C, and Nm23-H1, and two of the down-regulated proteins, Prx II and TrpRS, was examined in further detail. Analysis of mRNA expression levels demonstrated both transcription-dependent and transcription-independent regulation among the identified targets. Thus, this study reveals protein targets of p53 and highlights the role of transcription-independent effects for the p53-induced biological response.

  • 19. Roblick, U J
    et al.
    Hirschberg, Daniel
    Habermann, J K
    Palmberg, C
    Becker, S
    Krüger, S
    Gustafsson, M
    Bruch, H-P
    Franzén, B
    Ried, T
    Bergmann, T
    Auer, G
    Jörnvall, H
    Sequential proteome alterations during genesis and progression of colon cancer.2004In: Cellular and Molecular Life Sciences (CMLS), ISSN 1420-682X, E-ISSN 1420-9071, Vol. 61, no 10Article in journal (Refereed)
    Abstract [en]

    Changes in the proteome of colon mucosal cells accompany the transition from normal mucosa via adenoma and invasive cancer to metastatic disease. Samples from 15 patients with sporadic sigmoid cancers were analyzed. Proteins were separated by two-dimensional gel electrophoresis. Relative differences in expression levels between normal tissue, adenoma, carcinoma and metastasis were evaluated in both intra- and inter-patient comparisons. Up- and down-regulated proteins (> twofold) during development to cancer or metastasis were excised and submitted to peptide mass fingerprinting and MS/MS sequence analysis, facilitated by the use of a compact disc workstation. In total, 112 protein spots were found to be differentially regulated, of which 72 were determined as to protein identity, 46 being up-regulated toward the progression of cancer, and 26 down-regulated. Several of the identifications correlate with proteins of the cell cycle, cytoskeleton or metabolic pathways. The pattern changes now identified have the potential for design of marker panels for assistance in diagnostics and therapeutic strategies in colorectal cancer.

  • 20. Singh, I N
    et al.
    Goody, R J
    Goebel, S M
    Martin, K M
    Knapp, P E
    Marinova, Z
    Hirschberg, Daniel
    Yakovleva, T
    Bergman, T
    Bakalkin, G
    Hauser, K F
    Dynorphin A (1-17) induces apoptosis in striatal neurons in vitro through alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate receptor-mediated cytochrome c release and caspase-3 activation.2003In: Neuroscience, ISSN 0306-4522, E-ISSN 1873-7544, Vol. 122, no 4Article in journal (Refereed)
    Abstract [en]

    Dynorphin A (1-17), an endogenous opioid neuropeptide, can have pathophysiological consequences at high concentrations through actions involving glutamate receptors. Despite evidence of excitotoxicity, the basic mechanisms underlying dynorphin-induced cell death have not been explored. To address this question, we examined the role of caspase-dependent apoptotic events in mediating dynorphin A (1-17) toxicity in embryonic mouse striatal neuron cultures. In addition, the role of opioid and/or glutamate receptors were assessed pharmacologically using dizocilpine maleate (MK(+)801), a non-equilibrium N-methyl-D-aspartate (NMDA) antagonist; 6-cyano-7-nitroquinoxaline-2,3-dione, a competitive alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate antagonist; or (-)-naloxone, a general opioid antagonist. The results show that dynorphin A (1-17) (>or=10 nM) caused concentration-dependent increases in caspase-3 activity that were accompanied by mitochondrial release of cytochrome c and the subsequent death of cultured mouse striatal neurons. Moreover, dynorphin A-induced neurotoxicity and caspase-3 activation were significantly attenuated by the cell permeable caspase inhibitor, caspase-3 inhibitor-II (z-DEVD-FMK), further suggesting an apoptotic cascade involving caspase-3. AMPA/kainate receptor blockade significantly attenuated dynorphin A-induced cytochrome c release and/or caspase-3 activity, while NMDA or opioid receptor blockade typically failed to prevent the apoptotic response. Last, dynorphin-induced caspase-3 activation was mimicked by the ampakine CX546 [1-(1,4-benzodioxan-6-ylcarbonyl)piperidine], which suggests that the activation of AMPA receptor subunits may be sufficient to mediate toxicity in striatal neurons. These findings provide novel evidence that dynorphin-induced striatal neurotoxicity is mediated by a caspase-dependent apoptotic mechanism that largely involves AMPA/kainate receptors.

  • 21. Trelle, Morten Beck
    et al.
    Hirschberg, Daniel
    Jansson, Anna
    Ploug, Michael
    Roepstorff, Peter
    Andreasen, Peter A
    Jørgensen, Thomas J D
    Hydrogen/deuterium exchange mass spectrometry reveals specific changes in the local flexibility of plasminogen activator inhibitor 1 upon binding to the somatomedin B domain of vitronectin.2012In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 51, no 41Article in journal (Refereed)
    Abstract [en]

    The native fold of plasminogen activator inhibitor 1 (PAI-1) represents an active metastable conformation that spontaneously converts to an inactive latent form. Binding of the somatomedin B domain (SMB) of the endogenous cofactor vitronectin to PAI-1 delays the transition to the latent state and increases the thermal stability of the protein dramatically. We have used hydrogen/deuterium exchange mass spectrometry to assess the inherent structural flexibility of PAI-1 and to monitor the changes induced by SMB binding. Our data show that the PAI-1 core consisting of β-sheet B is rather protected against exchange with the solvent, while the remainder of the molecule is more dynamic. SMB binding causes a pronounced and widespread stabilization of PAI-1 that is not confined to the binding interface with SMB. We further explored the local structural flexibility in a mutationally stabilized PAI-1 variant (14-1B) as well as the effect of stabilizing antibody Mab-1 on wild-type PAI-1. The three modes of stabilizing PAI-1 (SMB, Mab-1, and the mutations in 14-1B) all cause a delayed latency transition, and this effect was accompanied by unique signatures on the flexibility of PAI-1. Reduced flexibility in the region around helices B, C, and I was seen in all three cases, which suggests an involvement of this region in mediating structural flexibility necessary for the latency transition. These data therefore add considerable depth to our current understanding of the local structural flexibility in PAI-1 and provide novel indications of regions that may affect the functional stability of PAI-1.

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