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  • 3201.
    Zou, JY
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturell molekylärbiologi.
    Kleywegt, GJ
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturell molekylärbiologi.
    Stahlberg, J
    Driguez, H
    Nerinckx, W
    Claeyssens, M
    Koivula, A
    Teerii, TT
    Jones, TA
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturell molekylärbiologi.
    Crystallographic evidence for substrate ring distortion and protein conformational changes during catalysis in cellobiohydrolase Cel6A from Trichoderma reesei1999Ingår i: STRUCTURE WITH FOLDING & DESIGN, ISSN 0969-2126, Vol. 7, nr 9, s. 1035-1045Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    BACKGROUND: Cel6A is one of the two cellobiohydrolases produced by Trichoderma reesei. The catalytic core has a structure that is a variation of the classic TIM barrel. The active site is located inside a tunnel, the roof of which is formed mainly by a pair of loops. RESULTS: We describe three new ligand complexes. One is the structure of the wild-type enzyme in complex with a nonhydrolysable cello-oligosaccharide, methyl 4-S-beta-cellobiosyl-4-thio-beta-cellobioside (Glc)(2)-S-(Glc)(2), which differs from a cellotetraose in the nature of the central glycosidic linkage where a sulphur atom replaces an oxygen atom. The second structure is a mutant, Y169F, in complex with the same ligand, and the third is the wild-type enzyme in complex with m-iodobenzyl beta-D-glucopyranosyl-beta(1,4)-D-xylopyranoside (IBXG). CONCLUSIONS: The (Glc)(2)-S-(Glc)(2) ligand binds in the -2 to +2 sites in both the wild-type and mutant enzymes. The glucosyl unit in the -1 site is distorted from the usual chair conformation in both structures. The IBXG ligand binds in the -2 to +1 sites, with the xylosyl unit in the -1 site where it adopts the energetically favourable chair conformation. The -1 site glucosyl of the (Glc)(2)-S-(Glc)(2) ligand is unable to take on this conformation because of steric clashes with the protein. The crystallographic results show that one of the tunnel-forming loops in Cel6A is sensitive to modifications at the active site, and is able to take on a number of different conformations. One of the conformational changes disrupts a set of interactions at the active site that we propose is an integral part of the reaction mechanism.

  • 3202.
    Zubarev, Roman A.
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Nielsen, Michael L.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Fung, Eva M.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Savitski, Mikhail M.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Kel-Margoulis, Olga
    Wingender, Edgar
    Kel, Alexander
    Identification of dominant signaling pathways from proteomics expression data2008Ingår i: Journal of Proteomics, ISSN 1874-3919, Vol. 71, nr 1, s. 89-96Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The availability of the results of high-throughput analyses coming from 'omic' technologies has been one of the major driving forces of pathway biology. Analytical pathway biology strives to design a 'pathway search engine', where the input is the 'omic' data and the output is the list of activated or dominant pathways in a given sample. Here we describe the first attempt to design and validate such a pathway search engine using as input expression proteomics data. The engine represents a specific workflow in computational tools developed originally for mRNA analysis (BMC Bioinformatics 2006, 7 (Suppl 2), S13). Using our own datasets as well as data from recent proteomics literature we demonstrate that different dominant pathways (EGF, TGF(beta), stress, and Fas pathways) can be correctly identified even from limited datasets. Pathway search engines can find application in a variety of proteomics-related fields, from fundamental molecular biology to search for novel types of disease biomarkers.

  • 3203.
    Zubarev, Roman A
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Zubarev, Alexander R
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Savitski, Mikhail M
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Electron capture/transfer versus collisionally activated/induced dissociations: Solo or duet?2008Ingår i: Journal of the American Society for Mass Spectrometry, ISSN 1044-0305, E-ISSN 1879-1123, Vol. 19, nr 6, s. 753-761Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    New ion fragmentation technologies-electron capture dissociation (ECD) and electron-transfer dissociation (ETD)-are based on interaction of multiply charged polypeptides with either free electrons (ECD) or anionic species (ETD). After initial difficulties, these ECD/ETD (ExD) technologies are now being increasingly implemented in high-throughput proteornics work. This critical analysis presents arguments for the combined use of ExD with the conventional low-energy collisional excitation CID/CAD (CxD). It is argued that the database search, a key technology in MS/MS-based proteomics, is vulnerable with respect to the incomplete sequence information obtainable with either of the techniques, peptide MS/MS homology being a major complicating factor. De novo sequencing is viewed as the only adequate answer to this challenge and it can be achieved only with combined use of ExD and CxD. The payoff in the form of additional sequence information is projected to exceed the costs of such implementation. The greatest impact of combining ExD and CxD is expected in high-resolution instruments.

  • 3204.
    Zyubko, Tatyana
    et al.
    Skolkovo Inst Sci & Technol, Ctr Life Sci, Moscow, Russia; Peter Great St Petersburg Polytech Univ, St Petersburg, Russia.
    Serebryakova, Marina
    Skolkovo Inst Sci & Technol, Ctr Life Sci, Moscow, Russia; Lomonosov Moscow State Univ, AN Belozersky Inst Physicochem Biol, Moscow Russia; Russian Acad Sci, Inst Gene Biol, Moscow, Russia.
    Andreeva, Julia
    Skolkovo Inst Sci & Technol, Moscow, Russia; Russian Acad Sci, Moscow, Russia.
    Metelev, Mikhail
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Molekylär systembiologi. Skolkovo Inst Sci & Technol, Moscow, Russia; Peter Great St Petersburg Polytech Univ, St Petersburg, Russia; Russian Acad Sci, Inst Gene Biol, Moscow Russia.
    Lippens, Guy
    Ups, INSA, INRA, TBI, CNRS, Toulouse, France.
    Dubiley, Svetlana
    Skolkovo Inst Sci & Technol, Moscow, Russia; Russian Acad Sci, Inst Gene Biol, Moscow, Russia.
    Severinov, Konstantin
    Skolkovo Inst Sci & Technol, Moscow, Russia; Waksman Inst Microbiol, Piscataway, NJ USA.
    Efficient in vivo synthesis of lasso peptide pseudomycoidin proceeds in the absence of both the leader and the leader peptidase2019Ingår i: Chemical Science, ISSN 2041-6520, E-ISSN 2041-6539, Vol. 10, nr 42, s. 9699-9707Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Bacterial lasso peptides are made from linear ribosomally synthesized precursors by specific cleavage at the leader-core junction site of the precursor by a dedicated protease recognizing the leader, followed by cyclisation of the newly formed N-terminus of the core part with a side chain of the internal aspartic or glutamic residue catalyzed by a macrolactam synthetase. The resulting structure has a tail that is threaded and fixed inside the cycle formed. Here, we characterize a new lasso peptide, pseudomycoidin, encoded by Bacillus pseudomycoides DSM 12442. The most surprising and unique feature of pseudomycoidin is that it can be produced in vivo from the ribosomally synthesized core part by a macrolactam synthetase, in the absence of the leader protease. The minimalism of the pseudomycoidin synthesis system makes it a powerful model to generate pseudomycoidin-based lasso-peptide libraries and to study the poorly understood process of lasso formation. We detected two additional pseudomycoidin modifications: phosphorylation of a terminal residue that was previously observed in another lasso peptide, followed by glycosylation, which was not observed heretofore. We speculate that these bulky C-terminal modifications may help maintain the threaded lasso topology of the compound synthesized by the macrolactam synthetase.

  • 3205.
    Álvarez-Castro, José M.
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Centrum för bioinformatik.
    Carlborg, Örjan
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Centrum för bioinformatik.
    A unified model for functional and statistical epistasis and its application in quantitative trait loci analysis2007Ingår i: Genetics, ISSN 0016-6731, E-ISSN 1943-2631, Vol. 176, nr 2, s. 1151-1167Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Interaction between genes, or epistasis, is found to be common and it is a key, concept for understanding adaptation and evolution of natural populations, response to selection in breeding programs, and determination of complex disease. Current]),, two independent classes of models are used to study epistasis. Statistical models focus on maintaining desired statistical properties for detection and estimation of genetic effects and for the decomposition of genetic variance using average effects of allele Substitutions in populations as parameters. Functional models focus on the evolutionary consequences of the attributes of the genotype-phenotype map using natural effects of allele substitutions as parameters. Here we provide a new, general and unified model framework: the natural and orthogonal interactions (NOIA) model. NOIA implements tools for transforming genetic effects measured in One Population to the ones of other populations (e.g., between two experimental designs for QTL) and parameters of statistical and functional epistasis into each other (thus enabling us to obtain functional estimates of QTL), as demonstrated numerically. We develop graphical interpretations of functional and statistical models as regressions of the genotypic values on the gene content, which illustrates the difference between the models-the constraint on the slope of the functional regression-and when the models are equivalent. Furthermore, we use our theoretical foundations to conceptually clarify functional and statistical epistasis, discuss the advantages of NOIA over previous theory, and stress the importance of linking functional and statistical models.

  • 3206.
    Ástvaldsson, Ásgeir
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Pathogenesis and Cell Biology of the Salmon Parasite Spironucleus salmonicida2019Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Spironucleus species are classified as diplomonad organisms, diverse eukaryotic flagellates found in oxygen-deprived environments. Members of Spironucleus are parasitic and can infect a variety of hosts, such as mice and birds, while the majority are found to infect fish. Massive outbreaks of severe systemic infection caused by a Spironucleus member, Spironucleus salmonicida (salmonicida = salmon killer), have been reported in farmed salmonids resulting in large economic impacts for aquaculture.

    In this thesis, the S. salmonicida genome was sequenced and compared to the genome of its diplomonad relative, the mammalian pathogen G. intestinalis (Paper I). Our analyses revealed large genomic differences between the two parasites that collectively suggests that S. salmonicida is more capable of adapting to different environments. As S. salmonicida can infiltrate different host tissues, we provide molecular evidence for how the parasite can tolerate oxygenated environments and suggest oxygen as a potential regulator of virulence factors (Paper III). To further investigate the molecular responses of the parasite and in addition, its host, during infection we set up an interaction system of S. salmonicida and ASK (Atlantic salmon kidney) cells (Paper VI).

    To study the cell biology in S. salmonicida we optimized an enzymatic proximity labeling method using ascorbate peroxidase (APEX) as a reporter for transmission electron microscopy (TEM) (Paper IV). As the system is robust and versatile, we showed the localization and performed ultrastructural characterization of numerous proteins in S. salmonicida and G. intestinalis. We furthermore utilized the APEX system to study the annexin protein family in S. salmonicida (Paper II). Super resolution microscopy and TEM were applied to show that the annexins are mostly associated with cytoskeletal and membranous structures. In addition, we performed phylogenetic analyses concluding that the annexin gene family is expanded in diplomonads.

    We performed experimental infection in Atlantic salmon and derived a potential model for the route of infection (Paper V). The results suggested multiple routes of transmission between hosts for the parasite.

    To conclude, the comprehensive work in this thesis has provided valuable insights into the pathogenesis and cell biology of the highly adaptable diplomonad parasite S. salmonicida.      

    Delarbeten
    1. The genome of Spironucleus salmonicida highlights a fish pathogen adapted to fluctuating environments
    Öppna denna publikation i ny flik eller fönster >>The genome of Spironucleus salmonicida highlights a fish pathogen adapted to fluctuating environments
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    2014 (Engelska)Ingår i: PLoS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 10, nr 2, s. e1004053-Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Spironucleus salmonicida causes systemic infections in salmonid fish. It belongs to the group diplomonads, binucleated heterotrophic flagellates adapted to micro-aerobic environments. Recently we identified energy-producing hydrogenosomes in S. salmonicida. Here we present a genome analysis of the fish parasite with a focus on the comparison to the more studied diplomonad Giardia intestinalis. We annotated 8067 protein coding genes in the ∼12.9 Mbp S. salmonicida genome. Unlike G. intestinalis, promoter-like motifs were found upstream of genes which are correlated with gene expression, suggesting a more elaborate transcriptional regulation. S. salmonicida can utilise more carbohydrates as energy sources, has an extended amino acid and sulfur metabolism, and more enzymes involved in scavenging of reactive oxygen species compared to G. intestinalis. Both genomes have large families of cysteine-rich membrane proteins. A cluster analysis indicated large divergence of these families in the two diplomonads. Nevertheless, one of S. salmonicida cysteine-rich proteins was localised to the plasma membrane similar to G. intestinalis variant-surface proteins. We identified S. salmonicida homologs to cyst wall proteins and showed that one of these is functional when expressed in Giardia. This suggests that the fish parasite is transmitted as a cyst between hosts. The extended metabolic repertoire and more extensive gene regulation compared to G. intestinalis suggest that the fish parasite is more adapted to cope with environmental fluctuations. Our genome analyses indicate that S. salmonicida is a well-adapted pathogen that can colonize different sites in the host.

    Nationell ämneskategori
    Mikrobiologi Genetik
    Identifikatorer
    urn:nbn:se:uu:diva-224545 (URN)10.1371/journal.pgen.1004053 (DOI)000332021500041 ()24516394 (PubMedID)
    Tillgänglig från: 2014-05-14 Skapad: 2014-05-14 Senast uppdaterad: 2019-03-19Bibliografiskt granskad
    2. Comparative cell biology and evolution of Annexins in Diplomonads
    Öppna denna publikation i ny flik eller fönster >>Comparative cell biology and evolution of Annexins in Diplomonads
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    2016 (Engelska)Ingår i: MSphere, ISSN 2379-5042, Vol. 1, nr 2, artikel-id e00032-15Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Annexins are multifunctional, calcium-binding proteins found in organisms across all kingdoms. Most studies of annexins from single-celled eukaryotes have focused on the alpha-giardins, proteins assigned to the group E annexins, expressed by the diplomonad Giardia intestinalis. We have characterized the annexin gene family in another diplomonad parasite, Spironucleus salmonicida, by phylogenetic and experimental approaches. We constructed a comprehensive phylogeny of the diplomonad group E annexins and found that they are abundant across the group with frequent gene duplications and losses. The annexins of S. salmonicida were found to be related to alpha-giardins but with better-preserved type II Ca2+ coordination sites. Two annexins were confirmed to bind phospholipids in a Ca2+-dependent fashion but with different specificities. Superresolution and confocal microscopy of epitope-tagged S. salmonicida annexins revealed localization to distinct parts of the cytoskeleton and membrane. The ultrastructural details of the localization of several annexins were determined by proximity labeling and transmission electron microscopy. Two annexins localize to a novel cytoskeletal structure in the anterior of the cell. Our results show that the annexin gene family is expanded in diplomonads and that these group E annexins are associated mostly with cytoskeletal and membrane structures. IMPORTANCE Annexins are proteins that associate with phospholipids in a Ca2+-dependent fashion. These proteins have been intensely studied in animals and plants because of their importance in diverse cellular processes, yet very little is known about annexins in single-celled eukaryotes, which represent the largest diversity of organisms. The human intestinal parasite Giardia intestinalis is known to have more annexins than humans, and they contribute to its pathogenic potential. In this study, we investigated the annexin complement in the salmon pathogen Spironucleus salmonicida, a relative of G. intestinalis. We found that S. salmonicida has a large repertoire of annexins and that the gene family has expanded separately across diplomonads, with members showing sequence diversity similar to that seen across kingdom-level groups such as plants and animals. S. salmonicida annexins are prominent components of the cytoskeleton and membrane. Two annexins are associated with a previously unrecognized structure in the anterior of the cell.

    Ort, förlag, år, upplaga, sidor
    Uppsala: , 2016
    Nyckelord
    intestinal parasite, annexins, diplomonad, Spironucleus salmonicida, Giardia, proximity labeling, APEX
    Nationell ämneskategori
    Cellbiologi Mikrobiologi Evolutionsbiologi
    Identifikatorer
    urn:nbn:se:uu:diva-264537 (URN)10.1128/mSphere.00032-15 (DOI)000392584700008 ()
    Forskningsfinansiär
    Vetenskapsrådet, 2012-3364Forskningsrådet Formas, 2013-910
    Tillgänglig från: 2015-10-14 Skapad: 2015-10-14 Senast uppdaterad: 2019-03-19Bibliografiskt granskad
    3. Oxygen induces the expression of invasion and stress response genes in the anaerobic salmon parasite Spironucleus salmonicida
    Öppna denna publikation i ny flik eller fönster >>Oxygen induces the expression of invasion and stress response genes in the anaerobic salmon parasite Spironucleus salmonicida
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    2019 (Engelska)Ingår i: BMC Biology, ISSN 1741-7007, E-ISSN 1741-7007, Vol. 17, nr 1, artikel-id 19Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Background: Spironucleus salmonicida is an anaerobic parasite that can cause systemic infections in Atlantic salmon. Unlike other diplomonad parasites, such as the human pathogen Giardia intestinalis, Spironucleus species can infiltrate the blood stream of their hosts eventually colonizing organs, skin and gills. How this presumed anaerobe can persist and invade oxygenated tissues, despite having a strictly anaerobic metabolism, remains elusive.

    Results: To investigate how S. salmonicida response to oxygen stress, we performed RNAseq transcriptomic analyses of cells grown in the presence of oxygen or antioxidant-free medium. We found that over 20% of the transcriptome is differentially regulated in oxygen (1705 genes) and antioxidant-depleted (2280 genes) conditions. These differentially regulated transcripts encode proteins related to anaerobic metabolism, cysteine and Fe-S cluster biosynthesis, as well as a large number of proteins of unknown function. S. salmonicida does not encode genes involved in the classical elements of oxygen metabolism (e.g., catalases, superoxide dismutase, glutathione biosynthesis, oxidative phosphorylation). Instead, we found that genes encoding bacterial-like oxidoreductases were upregulated in response to oxygen stress. Phylogenetic analysis revealed some of these oxygen-responsive genes (e.g., nadh oxidase, rubrerythrin, superoxide reductase) are rare in eukaryotes and likely derived from lateral gene transfer (LGT) events into diplomonads from prokaryotes. Unexpectedly, we observed that many host evasion- and invasion-related genes were also upregulated under oxidative stress suggesting that oxygen might be an important signal for pathogenesis.

    Conclusion: While oxygen is toxic for related organisms, such as G. intestinalis, we find that oxygen is likely a gene induction signal for host invasion- and evasion-related pathways in S. salmonicida. These data provide the first molecular evidence for how S. salmonicida could tolerate oxic host environments and demonstrate how LGT can have a profound impact on the biology of anaerobic parasites.

    Nyckelord
    Anaerobiosis, Diplomonads, Giardia, Lateral gene transfer, Oxygen stress, Parasitology, Protist, RNAseq, Spironucleosis, Spironucleus
    Nationell ämneskategori
    Biokemi och molekylärbiologi
    Identifikatorer
    urn:nbn:se:uu:diva-378917 (URN)10.1186/s12915-019-0634-8 (DOI)000459961200001 ()
    Forskningsfinansiär
    EU, Europeiska forskningsrådet, 310039-PUZZLE_CELLStiftelsen för strategisk forskning (SSF)Vetenskapsrådet, 2015-04959Forskningsrådet Formas, 2016-00539
    Tillgänglig från: 2019-03-12 Skapad: 2019-03-12 Senast uppdaterad: 2019-03-22Bibliografiskt granskad
    4. Proximity Staining using Enzymatic Protein Tagging in Diplomonads
    Öppna denna publikation i ny flik eller fönster >>Proximity Staining using Enzymatic Protein Tagging in Diplomonads
    2019 (Engelska)Ingår i: mSphere, E-ISSN 2379-5042, Vol. 4, nr 2, artikel-id e00153-19Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    The diplomonads are a group of understudied eukaryotic flagellates whose most prominent member is the human pathogen Giardia intestinalis. Methods commonly used in other eukaryotic model systems often require special optimization in diplomonads due to the highly derived character of their cell biology. We have optimized a proximity labeling protocol using pea ascorbate peroxidase (APEX) as a reporter for transmission electron microscopy (TEM), to enable study of ultrastructural cellular details in diplomonads. Currently available TEM-compatible tags requires light-induced activation (1, 2) or are inactive in many cellular compartments (3) while ascorbate peroxidase has not been shown to have those limitations. Here we have optimized the in vivo activity of two versions of pea ascorbate peroxidase (APXW41F and APEX) using the diplomonad fish parasite Spironucleus salmonicida, a relative of G. intestinalis. We exploited the well-known peroxidase substrates, Amplex UltraRed and 3,3’-diaminobenzidine (DAB), to validate the activity of the two tags and argue that APEX is the most stable version to use in Spironucleus salmonicida. Next, we fused APEX to proteins with established localization to evaluate the activity of APEX in different cellular compartments of the diplomonad cell and used Amplex UltraRed as well as antibodies along with super-resolution microscopy to confirm the protein-APEX localization. The ultrastructural details of protein-APEX fusions were determined by TEM and we observed marker activity in all cellular compartments tested when using the DAB substrate. Finally, we show that the optimized conditions established for S. salmonicida can be used in the related diplomonad G. intestinalis.

    Nationell ämneskategori
    Cellbiologi
    Identifikatorer
    urn:nbn:se:uu:diva-379669 (URN)
    Forskningsfinansiär
    Forskningsrådet Formas, 2016-00539
    Tillgänglig från: 2019-03-19 Skapad: 2019-03-19 Senast uppdaterad: 2019-03-20
    5. Experimental challenge of Atlantic salmon (Salmo salar) with the diplomonad parasite Spironucleus salmonicida to characterize the infection cycle
    Öppna denna publikation i ny flik eller fönster >>Experimental challenge of Atlantic salmon (Salmo salar) with the diplomonad parasite Spironucleus salmonicida to characterize the infection cycle
    (Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Experimental infections were performed of Atlantic salmon (Salmo salar) from the Baltic Sea region with the Diplomonad fish parasite Spironucleus salmonicida in order to define the infection cycle, specifically the time-line and putative routes of transmission. An oral infection protocol using axenic parasites was developed, as were new diagnostic tools using PCR and specific antibodies. We also produced firefly luciferase expressing S. salmonicida parasites that could be identified in the infected fish using in vivo and ex vivo imaging. The new tools made it possible to follow the S. salmonicida infection cycle in detail. Three different stages of the infection were identified: one initial intestinal stage, followed by a blood stage and a final tissue stage. Parasites intubated into the intestine attached to the intestinal surface and were identified in the blood after 1-3 weeks. Skin lesions and infections of the muscles, internal organs and eyes were seen 4-10 weeks after initiation of infection. Several morphologically different forms of S. salmonicida cells were detected in ex vivo cell-cultures of biopsies from skin lesions. By this infection trial we have been able to show that S. salmonicida may use several alternative routes of transmission. One alternative is the fecal-oral route, similar to other Diplomonad parasites but the parasites can also be excreted directly into the surrounding water from the mucous layer of the skin or from an ulcerated skin lesion. This information can be used to prevent the transmission of the parasite in fish farms.

    Nyckelord
    Diplomonads, Spironucleus salmonis, Salmo salar, fish infection, pathology
    Nationell ämneskategori
    Mikrobiologi
    Identifikatorer
    urn:nbn:se:uu:diva-378925 (URN)
    Forskningsfinansiär
    Forskningsrådet Formas, 2016-00539
    Tillgänglig från: 2019-03-09 Skapad: 2019-03-09 Senast uppdaterad: 2019-03-19
    6. Dual transcriptomic analysis of Spironucleus salmonicida-infected salmon cells identifies putative virulence factors and host responses
    Öppna denna publikation i ny flik eller fönster >>Dual transcriptomic analysis of Spironucleus salmonicida-infected salmon cells identifies putative virulence factors and host responses
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    (Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
    Nationell ämneskategori
    Biokemi och molekylärbiologi
    Identifikatorer
    urn:nbn:se:uu:diva-379666 (URN)
    Tillgänglig från: 2019-03-19 Skapad: 2019-03-19 Senast uppdaterad: 2019-03-19
  • 3207.
    Åbrink, M
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi. MOLECULAR IMMUNOLOGY, LARS HELLMAN.
    Larsson, E
    Gobl, A
    Hellman, L
    Expression of lactoferrin in the kidney; implications for innate immunity and iron metabolism.2000Ingår i: Kidney International, Vol. 57, s. 2004-2010Artikel i tidskrift (Refereegranskat)
  • 3208.
    Åbrink, M
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi. MOLECULAR IMMUNOLOGY, LARS HELLMAN.
    Larsson, E
    Hellman, L
    Demethylation of ERV3, an endogenous retrovirus regulating the krüppel-related zinc finger gene H-plk, in several human cell lines arrested during early monocyte development.1998Ingår i: DNA and Cell Biol., Vol. 17, s. 27-37Artikel i tidskrift (Refereegranskat)
  • 3209.
    Åbrink, M
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi. MOLECULAR IMMUNOLOGY, LARS HELLMAN.
    Ortiz, JA
    Mark, C
    Sanchez, C
    Looman, C
    Hellman, L
    Chambon, P
    Losson, R
    Conserved interaction between distinct Kruppel-associated box domains and the transcriptional intermediary factor 1 beta2001Ingår i: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, ISSN 0027-8424, Vol. 98, nr 4, s. 1422-1426Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The Kruppel-associated box (KRAB) domain, originally identified as a 75-aa sequence present in numerous Kruppel-type zinc-finger proteins, is a potent DNA-binding-dependent transcriptional repression domain that is believed to function through interaction

  • 3210. Åbrink, Magnus
    et al.
    Ortiz, J.A.
    Mark, Charlotta
    Sanchez, C.
    Looman, Camilla
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Hellman, Lars
    Chambon, P.
    Losson, R.
    Conserved interaction between distinct Krüppel-associated box domains and the transcriptional intermediary factor 1 beta2001Ingår i: PNAS, Vol. 98, s. 1422-1426Artikel i tidskrift (Refereegranskat)
  • 3211. Åbrink, Magnus
    et al.
    Ortiz, José A.
    Mark, Charlotta
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Sanchez, Cecilia
    Looman, Camilla
    Hellman, Lars
    Chambon, Pierre
    Losson, Régine
    Conserved Interaction Between Distinct Krüppel-associated box Domains and the Transcriptional Intermediary factor 1β2001Ingår i: Procl. Natl. Acad. Sci. USA, Vol. 98, nr 4, s. 1422-1426Artikel i tidskrift (Refereegranskat)
  • 3212.
    Ågren, Josefin
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi.
    Structural and Functional Studies of Giant Proteins in Lactobacillus kunkeei2019Självständigt arbete på avancerad nivå (yrkesexamen), 20 poäng / 30 hpStudentuppsats (Examensarbete)
    Abstract [en]

    Lactobacillus kunkeei is one of the most abundant bacteria within the honey crop of the honey bee. Genome sequencing of L. kunkeei isolated from honey bees all over the world showed several genes unique for L. kunkeei. Among these orphan genes, an array of four to five highly conserved genes coding for giant extracellular proteins were found. Cryogenic electron microscopy imaging of a giant-protein preparation from L. kunkeei A00901 showed an overall structure similar to a long string with a knot at the end. Further analysis showed high similarity between the different giants at the N-terminus, and secondary structure predictions showed that the same region was rich in β-sheets.  These results, combined with the knowledge of other large extracellular proteins, led to the hypothesis that the “knot” domain is located at the N-terminus and that these proteins are used by the cell to latch on to the intestine lining or other cells in the honey crop.

    In this study, predictions were made to locate the N-terminal domains of two of these giant proteins. Four different constructs were made for each protein, where three constructs were designed for expression and purification of the N-terminal domain with different end-positions, and one construct was for a predicted β-solenoid domain located downstream from the N-terminal domain. The protein constructs were recombinantly produced in E. coli, and three of the N-terminal constructs from both proteins were purified. Thermal stability was tested using nano differential scanning fluorimetry (nanoDSF), Thermofluor, and circular dichroism (CD), which all showed characteristic melting curves at low melting temperatures, ranging from 33 °C to 44 °C, for all three constructs. During CD measurements, all three constructs showed refolding after thermal denaturation and a higher abundance of antiparallel β-sheets over α-helices. Looking at the protein structure, small angle X-ray scattering data indicated that all three proteins formed elongated structures. These results indicate that a folded domain has been found for both proteins. Although, further analysis will be required to determine the boundaries of the N-terminal domains, and to elucidate if these domains have anything to do with ligand binding and the L. kunkeei ability to latch onto the honey crop.

    Publikationen är tillgänglig i fulltext från 2022-08-25 16:25
  • 3213.
    Åkerberg, Helena
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för neurovetenskap.
    Fällmar, Helena
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för neurovetenskap.
    Sjödin, Paula
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för neurovetenskap.
    Boukharta, Lars
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Gutierrez-de-Teran, Hugo
    Lundell, Ingrid
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för neurovetenskap.
    Mohell, Nina
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för neurovetenskap.
    Larhammar, Dan
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för neurovetenskap.
    Mutagenesis of human neuropeptide Y/peptide YY receptor Y2 reveals additional differences to Y1 in interactions with highly conserved ligand positions2010Ingår i: Regulatory Peptides, ISSN 0167-0115, E-ISSN 1873-1686, Vol. 163, nr 1-3, s. 120-129Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Neuropeptide Y (NPY) and peptide YY (PYY) share similar to 70% of their 36 amino acids and bind to the same three human receptor subtypes, Y1, Y2 and Y5, even though these receptors only share similar to 30% sequence identity Based on our previous investigation of human Y1 we describe here a mutagenesis study of three corresponding positions in human Y2, i e Tyr(2 64), Val(6 58) and Tyr(7 31) Pharmacological characterization was performed with the four peptide agonists PYY, NPY, PYY(3-36) and NPY(13-36) as well as the non-peptide antagonist BIIE0246 Results from mutants where Tyr(2 64) has been substituted by Ala suggest that Tyr(2 64) is involved in the interaction with all investigated ligands whereas position Tyr(7 31) seems to be more important for interaction with the truncated peptide PYY(3-36) than with intact NPY Surprisingly, substitution of Tyr(7 31) with His, the corresponding residue in Y1, resulted in total loss of binding of iodinated porcine PYY The third position. Val(6 58), did not influence binding of any of the ligands. These findings differ from those obtained for Y1 where Ala substitution resulted in lost or changed binding for each of the three positions. Although Tyr(2 64) and Tyr(7 31) in Y2 are involved in ligand binding, their interactions with the peptide ligands seem to be different from the corresponding positions in Y1 This suggests that the receptor-ligand interactions have changed during evolution after Y1 and Y2 arose from a common ancestral receptor.

  • 3214.
    Åkerbladh, Linda
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för läkemedelskemi, Avdelningen för organisk farmaceutisk kemi.
    Lu, Lu
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Konda, Konda
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för läkemedelskemi, Avdelningen för organisk farmaceutisk kemi.
    Cao, Sha
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Vocat, Anthony
    Maes, Louis
    Cole, Stewart T.
    Hughes, Diarmaid
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Larhed, Mats
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för läkemedelskemi, Avdelningen för organisk farmaceutisk kemi.
    Brandt, Peter
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för läkemedelskemi, Avdelningen för organisk farmaceutisk kemi.
    Karlén, Anders
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för läkemedelskemi, Avdelningen för organisk farmaceutisk kemi.
    Mowbray, Sherry
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Synthesis and in vitro biological evaluation of quinolinyl pyrimidines targeting type II NADH-dehydrogenase (NDH-2)Ingår i: Artikel i tidskrift (Övrigt vetenskapligt)
  • 3215.
    Åkerlund, T., Gullbrand, B., and Nordström, K.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi. MICROBIOLOGY.
    Effect of the Min system on nucleoid segregation in Escherichia coli2002Ingår i: Microbiology, Vol. 148, s. 3213-3222Artikel i tidskrift (Refereegranskat)
  • 3216.
    Åkerlund, Thomas, Gullbrand, Björn, and Nordström, Kurt
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Effects of the Min system on nucleoid segregation in Escherichia coli.2002Ingår i: Microbioology, Vol. 148, s. 3213-3222Artikel i tidskrift (Refereegranskat)
  • 3217.
    Åqvist, J.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Calculation of Absolute Binding Free Energies for Charged Ligands and the Effects of Long-Range Electrostatic Interactions1996Ingår i: J. Comput. Chem., Vol. 17, s. 1587-Artikel i tidskrift (Refereegranskat)
  • 3218.
    Åqvist, J.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Comment on Transferability of Ion Models1994Ingår i: J. Phys. Chem., Vol. 98, s. 8253-Artikel i tidskrift (Refereegranskat)
  • 3219.
    Åqvist, J.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Computer Modeling of Chemical Reactions in Enzymes and Solution1993Ingår i: J. Biochem. Biophys. Methods, Vol. 26, s. 241-Artikel, recension (Övrigt vetenskapligt)
  • 3220.
    Åqvist, J.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Long-Range Electrostatic Effects on Peptide Folding1999Ingår i: FEBS Lett., Vol. 457, s. 414-Artikel i tidskrift (Refereegranskat)
  • 3221.
    Åqvist, J.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Metalloenzymes Involving Amino Acid-Residue and Related Radicals1995Ingår i: Q. Rev. Biol., Vol. 70, s. 505-Artikel, recension (Övrigt vetenskapligt)
  • 3222.
    Åqvist, J.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Modelling of Proton Transfer Reactions in Enzymes1997Ingår i: Computational Approaches to Biochemical Reactivity, Kluwer, Dordrecht , 1997, s. 341-Kapitel i bok, del av antologi (Refereegranskat)
  • 3223.
    Åqvist, J.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    On the Sensitivity to Changes in Water-Protein Interaction Parameters1997Ingår i: Proteins, Vol. 28, s. 143-Artikel i tidskrift (Refereegranskat)
  • 3224.
    Åqvist, J. & Fothergill, M.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Computer Simulation of the Triosephosphate Isomerase Catalyzed Reaction1996Ingår i: J. Biol. Chem., Vol. 271, s. 10010-Artikel i tidskrift (Refereegranskat)
  • 3225.
    Åqvist, J. & Hansson, T.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Analysis of Electrostatic Potential Truncation Schemes in Simulations of Polar Solvents1998Ingår i: J. Phys. Chem., Vol. 102, s. 3837-Artikel i tidskrift (Refereegranskat)
  • 3226.
    Åqvist, J. & Hansson, T.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    On the Validity of Electrostatic Linear Response in Polar Solvents1996Ingår i: J. Phys. Chem., Vol. 100, s. 9512-Artikel i tidskrift (Refereegranskat)
  • 3227.
    Åqvist, J. & Luzhkov, V.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Ion Permeation Mechanism of the K+ Channel2000Ingår i: Nature, Vol. 404, s. 881-Artikel i tidskrift (Refereegranskat)
  • 3228.
    Åqvist, J. & Marelius, J.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    The Linear Interaction Energy Method for Computation of Ligand Binding Affinities2001Ingår i: Free Energy Calculations in Rational Drug Design, Kluwer, New York , 2001, s. 171-Kapitel i bok, del av antologi (Refereegranskat)
  • 3229.
    Åqvist, J. & Marelius, J.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    The Linear Interaction Energy Method for Predicting Ligand Binding Free Energies2001Ingår i: J. Combin. Chem. High Throughput Screening, Vol. 4, s. 613-Artikel i tidskrift (Refereegranskat)
  • 3230.
    Åqvist, J. & Warshel, A.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Molecular Recognition in the Catalytic Action of Metalloenzymes1993Ingår i: Principles of Molecular Recognition, Chapman & Hall , 1993, s. 108-Kapitel i bok, del av antologi (Refereegranskat)
  • 3231.
    Åqvist, J., Fothergill, M. & Warshel, A.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Computer Simulation of the CO2/HCO3- Interconversion Step in Human Carbonic Anhydrase I1993Ingår i: J. Am. Chem. Soc., Vol. 115, s. 631-Artikel i tidskrift (Refereegranskat)
  • 3232.
    Åqvist, J., Kolmodin, K., Florian, J. & Warshel, A.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Mechanistic Alternatives in Phosphate Ester Hydrolysis1999Ingår i: Chemistry & Biology, Vol. 6, s. R71-Artikel i tidskrift (Refereegranskat)
  • 3233.
    Åqvist, J., Luzhkov, V.B. & Brandsdal, B.O.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Ligand Binding Affinities from MD Simulations2002Ingår i: Acc. Chem. Res., Vol. 35, s. 358-Artikel i tidskrift (Refereegranskat)
  • 3234.
    Åqvist, J., Medina, C. & Samuelsson, J.-E.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    A New Method for Predicting Binding Affinity in Computer-Aided Drug Design1994Ingår i: Protein Eng., Vol. 7, s. 385-Artikel i tidskrift (Refereegranskat)
  • 3235.
    Åqvist, J.
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Warshel, A.
    Simulation of Enzyme Reactions Using Valence Bond Force Fields and Other Hybrid Quantum/Classical Approaches1993Ingår i: Chemical ReviewsArtikel i tidskrift (Refereegranskat)
  • 3236.
    Åqvist, J
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturell molekylärbiologi.
    Wennerström, P
    Nervall, M
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturell molekylärbiologi.
    Bjelic, S
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturell molekylärbiologi.
    Brandsdal, B
    Molecular Dynamics Simulations of Water and Biomolecules with a Monte Carlo Constant Pressure Algorithm2004Ingår i: Chem. Phys. Lett., Vol. 384, s. 288.-Artikel i tidskrift (Refereegranskat)
  • 3237.
    Åqvist, Johan
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Beräkningsbiologi och bioinformatik.
    Cold Adaptation of Triosephosphate Isomerase2017Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 56, nr 32, s. 4169-4176Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The main problem for enzymes from psychrophilic species, which need to work near the freezing point of liquid water, is the exponential decay of reaction rates as the temperature is decreased. Cold-adapted enzymes have solved this problem by shifting the activation enthalpy-entropy balance for the catalyzed reaction compared to those of their mesophilic orthologs. To understand the structural basis of this universal feature, it is necessary to examine pairs of such orthologous enzymes, with known three-dimensional structures, at the microscopic level. Here, we use molecular dynamics free energy calculations in combination with the empirical valence bond method to evaluate the temperature dependence of the activation free energy for differently adapted triosephosphate isomerases. The results show that the enzyme from the psychrophilic bacterium Vibrio marinus indeed displays the characteristic shift in enthalpy-entropy balance, compared to that of the yeast ortholog. The origin of this effect is found to be located in a few surface-exposed protein loops that show differential mobilities in the two enzymes. Key mutations render these loops more mobile in the cold-adapted triosephosphate isomerase, which explains both the reduced activation enthalpy contribution from the protein surface and the lower thermostability.

  • 3238.
    Åqvist, Johan
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Beräkningsbiologi och bioinformatik.
    Computing the temperature dependence of chemical reactions inside and outside of living things2015Ingår i: European Biophysics Journal, ISSN 0175-7571, E-ISSN 1432-1017, Vol. 44, s. S160-S160Artikel i tidskrift (Övrigt vetenskapligt)
  • 3239.
    Åqvist, Johan
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Beräkningsbiologi och bioinformatik.
    Isaksen, Geir Villy
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Beräkningsbiologi och bioinformatik. Univ Tromso, Dept Chem, Ctr Theoret & Computat Chem, N-9037 Tromso, Norway..
    Brandsdal, Bjorn Olav
    Univ Tromso, Dept Chem, Ctr Theoret & Computat Chem, N-9037 Tromso, Norway..
    Computation of enzyme cold adaptation2017Ingår i: Nature Reviews Chemistry, E-ISSN 2397-3358, Vol. 1, nr 7, artikel-id UNSP 0051Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    Earth has several environments that are potentially hostile to life. The survival of organisms has required the expression of proteins that are adapted to function under extreme temperature, pH, pressure or ionic strength. However, the origin of such adaptations remains, in most cases, an open question. This Review presents a detailed analysis of the specialized enzymes that are able to maintain high catalytic rates at low temperatures and highlights the important role that computational studies have in uncovering the evolutionary principles behind the cold adaptation of enzymes. Although often highly homologous to their mesophilic counterparts, these cold-adapted enzymes have characteristic and universal properties that reflect their evolutionary optimization. In addition to exhibiting maximum reaction rates at lower temperatures, cold-adapted enzymes are more heat-labile and their catalytic mechanisms have distinct signatures in terms of the thermodynamic activation parameters. The structural origins of these properties have been elusive but are hypothesized to be related to protein flexibility.

  • 3240.
    Åqvist, Johan
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Beräknings- och systembiologi.
    Kamerlin, Lynn Shina Caroline
    The Conformation of a Catalytic Loop Is Central to GTPase Activity on the Ribosome2015Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 54, nr 2, s. 546-556Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The translational GTPases hydrolyze GTP on the ribosome at several stages of the protein synthesis cycle. Because of the strong conservation of their catalytic center, these enzymes are expected to operate through a universal hydrolysis mechanism, in which a critical histidine residue together with the sarcin-ricin loop of the large ribosomal subunit is necessary for GTPase activation. Here we examine different possible pathways for GTP hydrolysis by EF-Tu through extensive computer simulations. We show that a conformational change of the peptide plane preceding this histidine has a decisive effect on the energetics of the reaction. This transition was predicted earlier by us and has recently been confirmed experimentally. It is found to promote early proton transfer from water to the gamma-phosphate group of GTP, followed by nucleophilic attack by hydroxide ion. The calculated reaction energetics is in good agreement with available kinetic data, for both wild-type and mutant versions of EF-Tu, and indicates that the latter may enforce a change in mechanism toward more concerted pathways.

  • 3241.
    Åqvist, Johan
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Kazemi, Masoud
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Isaksen, Geir Villy
    Univ Tromso, Dept Chem, Ctr Theoret Computat Chem, N-9037 Tromso, Norway..
    Brandsdal, Bjorn Olav
    Univ Tromso, Dept Chem, Ctr Theoret Computat Chem, N-9037 Tromso, Norway..
    Entropy and Enzyme Catalysis2017Ingår i: Accounts of Chemical Research, ISSN 0001-4842, E-ISSN 1520-4898, Vol. 50, nr 2, s. 199-207Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    CONSPECTUS: The role played by entropy for the enormous rate enhancement achieved by enzymes has been debated for many decades. There are, for example, several confirmed cases where the activation free energy is reduced by around 10 kcal/mol due to entropic effects, corresponding to a rate enhancement of similar to 10(7) compared to the uncatalyzed reaction. However, despite substantial efforts from both the experimental and theoretical side, no real consensus has been reached regarding the origin of such large entropic contributions to enzyme catalysis. Another remarkable instance of entropic effects is found in enzymes that are adapted by evolution to work at low temperatures, near the freezing point of water. These cold-adapted enzymes invariably show a more negative entropy and a lower enthalpy of activation than their mesophilic orthologs, which counteracts the exponential damping of reaction rates at lower temperature. The structural origin of this universal phenomenon has, however, remained elusive. The basic problem with connecting macroscopic thermodynamic quantities, such as activation entropy and enthalpy derived from Arrhenius plots, to the 3D protein structure is that the underlying detailed (microscopic) energetics is essentially inaccessible to experiment. Moreover, attempts to calculate entropy contributions by computer simulations have mostly focused only on substrate entropies, which do not provide the full picture. We have recently devised a new approach for accessing thermodynamic activation parameters of both enzyme and solution reactions from computer simulations, which turns out to be very successful. This method is analogous to the experimental Arrhenius plots and directly evaluates the temperature dependence of calculated reaction free energy profiles. Hence, by extensive molecular dynamics simulations and calculations of up to thousands of independent free energy profiles, we are able to extract activation parameters with sufficient precision for making direct comparisons to experiment. We show here that the agreement with the measured quantities, for both enzyme catalyzed and spontaneous solution reactions, is quite remarkable. Importantly, we can now address some of the most spectacular entropy effects in enzymes and clarify their detailed microscopic origin. Herein, we discuss as examples the conversion of cytidine to uridine catalyzed by cytidine deaminase and reactions taking place on the ribosome, namely, peptide bond formation and GTP hydrolysis by elongation factor Tu. It turns out that the large entropy contributions to catalysis in these cases can now be rationalized by our computational approach. Finally, we address the problem of cold adaptation of enzyme reaction rates and prove by computational experiments that the universal activation enthalpy entropy phenomenon originates from mechanical properties of the outer protein surface.

  • 3242.
    Åström, Anders
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi, Medicinsk genetik.
    Åström, Jonas
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi, Medicinsk genetik.
    Virtanen, Anders
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi. Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi, Medicinsk genetik.
    A simple procedure for isolation of eukaryotic mRNA polyadenylation factors1991Ingår i: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 202, nr 3, s. 765-773Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    We have devised a simple chromatographic procedure which isolates five polyadenylation factors that are required for polyadenylation of eukaryotic mRNA. The factors were separated from each other by fractionation of HeLa cell nuclear extract in two consecutive chromatographic steps. RNA cleavage at the L3 polyadenylation site of human adenovirus 2 required at least four factors. Addition of adenosine residues required only two of these factors. The fractionation procedure separates two components that are both likely to be poly(A) polymerases. The candidate poly(A) polymerases were interchangeable and participated during both RNA cleavage and adenosine addition. They were discriminated from each other by chromatographic properties, heat sensitivity and divalent cation requirement. We have compared our data with published information and have been able to correlate the activities that we have isolated to previously identified polyadenylation factors. However, we have not been able to assign one of the candidate poly(A) polymerases to a previously identified poly(A) polymerase. This simple fractionation procedure can be used for generating an in vitro reconstituted system for polyadenylation within a short period of time.

  • 3243.
    Åström, Jonas
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi, Medicinsk genetik.
    Åström, Anders
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi, Medicinsk genetik.
    Virtanen, Anders
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi. Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi, Medicinsk genetik.
    In vitro deadenylation of mammalian mRNA by a HeLa cell 3' exonuclease1991Ingår i: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 10, nr 10, s. 3067-3071Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    We have identified a 3' exonuclease in HeLa cell extracts which deadenylates mammalian mRNA and leaves the mRNA body intact after poly(A) removal. Only homopolymeric adenosine tails located at the 3' end were efficiently removed by the exonuclease. The poly(A) removing activity did not require any specific sequences in the mRNA body either for poly(A) removal or for accumulation of the deadenylated mRNA. We conclude that the poly(A) removing activity is a 3' exonuclease since (i) reaction intermediates gradually lose the poly(A) tail, (ii) degradation is prevented by the presence of a cordycepin residue at the 3' end and (iii) RNAs having internally located poly(A) stretches are poor substrates for degradation. The possible involvement of the poly(A) removing enzyme in regulating mRNA translation and stability is discussed.

  • 3244.
    Åström, Jonas
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi, Medicinsk genetik.
    Åström, Anders
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi, Medicinsk genetik.
    Virtanen, Anders
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Properties of a HeLa cell 3' exonuclease specific for degrading poly(A) tails of mammalian mRNA.1992Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 267, nr 25, s. 18154-18159Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A HeLa cell 3'-exonuclease with properties of a mammalian mRNA poly(A) tail-removing enzyme has been characterized. The exonuclease shows high specificity for the poly(A) tail, and it is single strand-specific and requires a 3'-hydroxyl group for its activity. During degradation 5'-AMP is liberated as a product, and a 3'-OH group is left on the last adenosine residue of the remaining poly(A) tail. The activity is inhibited by 5'-AMP and can be competed by poly(A)-containing mRNA or poly(A). Based on these findings we propose a reaction pathway for poly(A) tail removal catalyzed by the HeLa cell poly(A) tail-specific 3' exonuclease.

  • 3245. Öhman, Johan
    et al.
    Jakobsson, Emma
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Källström, Ulla
    Elmblad, Annette
    Ansari, Akbar
    Kalderen, Christina
    Robertson, Elinor
    Danielsson, Eva
    Gustavsson, Anna-Lena
    Varadi, Andrea
    Ekblom, Jonas
    Holmgren, Erik
    Doverskog, Magnus
    Abrahamsen, Lars
    Nilsson, Joakim
    Production of a truncated soluble human semicarbazide-sensitive amine oxidase (SSAO) mediated by a GST-fusion protein secreted from HEK293 cellsArtikel i tidskrift (Refereegranskat)
  • 3246.
    Öhrngren, Per
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för läkemedelskemi, Avdelningen för organisk farmaceutisk kemi.
    Wu, Xiongyu
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för läkemedelskemi, Avdelningen för organisk farmaceutisk kemi.
    Persson, Magnus
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi.
    Ekegren, Jenny
    Wallberg, Hans
    Vrang, Lotta
    Rosenquist, Åsa
    Samuelsson, Bertil
    Unge, Torsten
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi.
    Larhed, Mats
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för läkemedelskemi, Avdelningen för organisk farmaceutisk kemi.
    Hiv-1 Protease Inhibitors with a Tertiary Alcohol Containing a Transition-State Mimic and Various P2/P1´ Substituents2011Ingår i: MedChemComm, ISSN 2040-2503, E-ISSN 2040-2511, Vol. 2, nr 8, s. 701-709Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Two series, including in total 18 novel HIV-1 protease inhibitors, comprising a tertiary alcohol as thetransition-state mimic have been synthesised and evaluated. Replacement of the previously used, butmetabolically unstable, indanol amide group with amino acid derived aliphatic P2–P3 moietiesprovided potent inhibitors with low Ki- and EC50-values (2.7 nM and 2.0 mM, respectively). The P10subunit was varied using 10 different aromatic and heteroaromatic substituents furnishing thecorresponding inhibitors with retained activity. Permeability and stability studies showed examples inthe same range as Atazanavir. X-Ray crystallographic analysis of two selected inhibitor enzyme cocomplexes(9a and 9d) supplied detailed structural information. The binding modes were compared tothose of Atazanavir and a previously reported indanol amide containing inhibitor (14). The novelinhibitors with an elongated P1' side chain enabled a previously unexploited edge-on interaction withPhe53/153. Exchange of the previously used indanol amide P2 moiety, with a tert-leucine derived P2–P3side chain, furnished small main chain displacements in the S2–S3 pocket. The methyl amide in the P3 position caused a 2 Å shift of the Arg8/108 in comparison to 14, indicating the flexibility of the proteaseactive site.

  • 3247.
    Örlén, Hanna
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Hughes, Diarmaid
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Weak mutators can drive the evolution of fluoroquinolone resistance in Escherichia coli2006Ingår i: Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, E-ISSN 1098-6596, Vol. 50, nr 10, s. 3454-3456Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Weak mutators are common among clinical isolates of Escherichia coli. We show that the relative mutation rate and the "evolvability of fluoroquinolone resistance" are related by a power law slope of 1.2 over 3 orders of magnitude. Thus, even weak mutators can drive the evolution of fluoroquinolone resistance under selection pressure.

  • 3248.
    Östberg, Linus J.
    et al.
    Karolinska Inst, Sci Life Lab, Dept Med Biochem & Biophys, Stockholm, Sweden..
    Persson, Bengt
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Beräkningsbiologi och bioinformatik. Karolinska Inst, Sci Life Lab, Dept Med Biochem & Biophys, Stockholm, Sweden..
    Höög, Jan-Olov
    Karolinska Inst, Dept Med Biochem & Biophys, Stockholm, Sweden..
    Computational studies of human class V alcohol dehydrogenase - the odd sibling2016Ingår i: BMC Biochemistry, ISSN 1471-2091, E-ISSN 1471-2091, Vol. 17, artikel-id 16Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: All known attempts to isolate and characterize mammalian class V alcohol dehydrogenase (class V ADH), a member of the large ADH protein family, at the protein level have failed. This indicates that the class V ADH protein is not stable in a non-cellular environment, which is in contrast to all other human ADH enzymes. In this report we present evidence, supported with results from computational analyses performed in combination with earlier in vitro studies, why this ADH behaves in an atypical way. Results: Using a combination of structural calculations and sequence analyses, we were able to identify local structural differences between human class V ADH and other human ADHs, including an elongated beta-strands and a labile a-helix at the subunit interface region of each chain that probably disturb it. Several amino acid residues are strictly conserved in class I-IV, but altered in class V ADH. This includes a for class V ADH unique and conserved Lys51, a position directly involved in the catalytic mechanism in other ADHs, and nine other class V ADH-specific residues. Conclusions: In this study we show that there are pronounced structural changes in class V ADH as compared to other ADH enzymes. Furthermore, there is an evolutionary pressure among the mammalian class V ADHs, which for most proteins indicate that they fulfill a physiological function. We assume that class V ADH is expressed, but unable to form active dimers in a non-cellular environment, and is an atypical mammalian ADH. This is compatible with previous experimental characterization and present structural modelling. It can be considered the odd sibling of the ADH protein family and so far seems to be a pseudoenzyme with another hitherto unknown physiological function.