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  • 1051.
    Yentrapalli, Venkata ramesh
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Chronic low-dose gamma radiation exposure induces premature senescence in human umbilical vein endothelial cellsManuskript (preprint) (Övrigt vetenskapligt)
  • 1052.
    Yentrapalli, Venkata Ramesh
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Mechanism behind the development of radiation-induced cardiovascular effects2012Licentiatavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Epidemiological studies on acutely and chronically exposed individuals indicate that ionising radiation could be a risk factor for cardiovascular diseases. Experimental data regarding radiation-induced cardiovascular late effects are limited and biological mechanisms behind these late effects are still unclear. The estimation of risks concerning low-dose rate and low-dose ionising radiation is still challenging due to lack of experimental evidence.

    The first paper of the thesis describes the radiation-induced protein alterations in cardiac tissue of total body irradiated mice. We use a label-free proteomic approach to identify altered proteins from formalin-fixed paraffin-embedded heart tissue. Comparison between the cardiac proteomes of control and irradiated mice indicates the respiratory chain, lipid metabolism and pyruvate metabolism pathways are affected. We suggest that these biological processes may play a vital role in radiation-induced cardiovascular diseases.

    In the second paper (manuscript) we hypothesized that chronic low-dose rate ionising radiation accelerates premature senescence in endothelial cells and that this may contribute to the radiation-induced cardiovascular diseases. We tested our hypothesis by systematically analysing the growth rate, the number of accumulated senescent cells and the protein expression profiles of the chronically exposed primary human umbilical vein endothelial cells until the cells entered senescence. Decrease in cumulative population doublings and increase in senescent-associated beta-galactosidase stained cells show that chronic exposure to radiation accelerates endothelial cells senescence. Our proteomic results suggest that the cytoskeletal organisation, cell-cell communication and adhesion, inflammation and carbohydrate metabolism were influenced by chronic exposure to radiation. A few deregulated proteins have previously been associated with replicative and stress-induced premature senescence. The results presented here provide new insights on radiation-induced premature senescence in endothelial cells and the changes in protein expression associated with this process.

  • 1053.
    Yousif, Rafat
    Stockholms universitet, Naturvetenskapliga fakulteten, Fysikum.
    Investigating the influence of water in lysozyme structure and dynamics using FT-IR and XRD2019Självständigt arbete på grundnivå (kandidatexamen), 10 poäng / 15 hpStudentuppsats (Examensarbete)
    Abstract [en]

    Water is “the matrix of life” for its fascinating properties. The well-known simple water molecule consists of one oxygen atom and two hydrogen atoms, covering most of planet earth’ssurface. It is the most studied element in science; however, its properties are still not fully understood. Another essential building block of life is proteins, which manifest naturally in aqueous environments. The protein activity is controlled by the protein folding process that is dependent on the surrounding environment. It is hypothesized that the hydrogen bond network of water plays an important role in the folding process. Here, we investigate the protein lysozyme in liquid water as well as in the crystalline state ice Ih, exploring various temperatures, using FT-IR and XRD. Our main finding is that a transition occurs at approximately T=210 K, indicative of the hypothesised protein dynamic “glass” transitionobserved by previous studies in supercooled water at similar temperatures.

  • 1054. Ytterberg, A. Jimmy
    et al.
    Zubarev, Roman A.
    Baumgarten, Thomas
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Post-translational targeting of a recombinant protein promotes its efficient secretion into the E. coli periplasmManuskript (preprint) (Övrigt vetenskapligt)
  • 1055.
    Yu, Simei
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    ATPase dependent and independent roles of Brahma in transcription and pre-mRNA processing2015Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    SWI/SNF is a chromatin-remodeling complex and Brahma (BRM) is the ATPase subunit of SWI/SNF. BRM regulates transcription by remodeling the nucleosomes at promoter regions. BRM is also associated with RNA and affects pre-mRNA processing together with other SWI/SNF subunits. In this thesis, I will discuss the roles of BRM in both transcription and pre-mRNA processing. In Paper I, we showed that BRM, as well as other SWI/SNF subunits SNR1 and MOR, affects the alternative processing of a subset of pre-mRNAs, as shown by microarray analysis. This observation was validated by RNAi experiments both in Drosophila S2 cells and in vivo. In Paper II, we characterized the trans-splicing of transcripts derived from the mod(mdg4) gene. RNA interference (RNAi) and overexpression experiments revealed that BRM regulates the trans-splicing of mod(mdg4)-RX in an ATPase independent manner. In Paper III, we analyzed the expression of two BRM-target genes identified in Paper I, CG44250 and CG44251. RNAi and overexpression experiments showed that the expression levels of these two genes were affected by BRM in a manner that is independent of its ATPase activity. Transcriptome analysis further proved that BRM affects gene expression both in ATPase dependent and independent manners. In Paper IV, we showed that BRM is present at the 3’-end of two analyzed genes, CG5174 and CG2051. BRM facilitates the recruitment of the cleavage and polyadenylation machinery to the cleavage sites through protein-protein interactions that do not require the ATPase activity of BRM. Morevoer, BRM promotes the cleavage of the CG5174 and CG2051 pre-mRNAs. To sum up, SWI/SNF plays important roles not only in transcription but also in pre-mRNA processing. To regulate transcription, BRM can either act as an ATPase-dependent chromatin remodeler or in a manner that does not involve ATPase activity. Additionally, BRM interacts with RNA-binding proteins to regulate the processing of a subset of pre-mRNAs, and this function of BRM is independent of its chromatin remodeling activity.

  • 1056.
    Yu, Simei
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Gàñez-Zapater, Antoni
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Jordán-Pla, Antonio
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Rolicka, Anna T.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Östlund Farrants, Ann-Kristin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Visa, Neus
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    A role for SWI/SNF in pre-mRNA 3’-end processingManuskript (preprint) (Övrigt vetenskapligt)
  • 1057.
    Yu, Simei
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Waldholm, Johan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Böhm, Stefanie
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Visa, Neus
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Brahma regulates a specific trans-splicing event at the mod(mdg4) locus of Drosophila melanogaster2014Ingår i: RNA Biology, ISSN 1547-6286, E-ISSN 1555-8584, Vol. 11, nr 2, s. 134-145Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The mod(mdg4) locus of Drosophila melanogaster contains several transcription units encoded on both DNA strands. The mod(mdg4) pre-mRNAs are alternatively spliced, and a very significant fraction of the mature mod(mdg4) mRNAs are formed by trans-splicing. We have studied the transcripts derived from one of the anti-sense regions within the mod(mdg4) locus in order to shed light on the expression of this complex locus. We have characterized the expression of anti-sense mod(mdg4) transcripts in S2 cells, mapped their transcription start sites and cleavage sites, identified and quantified alternatively spliced transcripts, and obtained insight into the regulation of the mod(mdg4) trans-splicing. In a previous study, we had shown that the alternative splicing of some mod(mdg4) transcripts was regulated by Brahma (BRM), the ATPase subunit of the SWI/SNF chromatin-remodeling complex. Here we show, using RNA interference and overexpression of recombinant BRM proteins, that the levels of BRM affect specifically the abundance of a trans-spliced mod(mdg4) mRNA isoform in both S2 cells and larvae. This specific effect on trans-splicing is accompanied by a local increase in the density of RNA polymerase II and by a change in the phosphorylation state of the C-terminal domain of the large subunit of RNA polymerase II. Interestingly, the regulation of the mod(mdg4) splicing by BRM is independent of the ATPase activity of BRM, which suggests that the mechanism by which BRM modulates trans-splicing is independent of its chromatin-remodeling activity.

  • 1058.
    Yu, Simei
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Waldholm, Johan
    Jordán-Pla, Antonio
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Källman, Thomas
    Östlund Farrants, Ann-Kristin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Visa, Neus
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    The ATPase-dependent and ATPase-independent functions of Brahma in transcription regulationManuskript (preprint) (Övrigt vetenskapligt)
  • 1059.
    Yu, Xin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Studies of polyglutamine expanded Ataxin-7 toxicity2015Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Spinocerebellar ataxia type 7 (SCA7) is an autosomal dominant inherited neurodegenerative disease for which there is no cure. SCA7 belongs to the group of polyglutamine disorders, which are all caused by the expansion of a polyglutamine tract in different disease proteins. Common toxic mechanisms have been proposed for polyglutamine diseases; however the exact pathological mechanism(s) are still unclear.

    The aim of this thesis was to identify and characterize the molecular mechanisms by which polyglutamine expansion in the ATXN7 protein cause SCA7 and how this can be counteracted. We found that mutant ATXN7 can be degraded by the ubiquitin proteasome system (UPS) and autophagy, the two main cellular degradation pathways. However aggregation stabilized the protein against degradation. Moreover, we found that mutant ATXN7 blocked the induction of autophagy by interfering with p53 and the ULK1-ATG13-FIP200 complex. Pharmacological stimulation of autophagy ameliorated aggregation, as well as toxicity.

    We also found that oxidative stress plays an important role in mutant ATXN7 toxicity and that the oxidative stress is generated by activation of NADPH oxidase 1 (NOX1) complexes. Furthermore, we showed that the increased NOX1 activity, together with polyQ expanded ATXN7 mediated disruption of the transcription factor p53, results in metabolic alterations in SCA7 cells. The expression of key p53 regulated metabolic proteins like AIF, TIGAR and GLUT1 was altered in SCA7 cells and resulted in reduced mitochondrial respiration, a higher dependence on glycolysis and reduced ATP levels.

    In summary, our data indicate that mutant ATXN7 mediated dysregulation of p53, resulting in autophagic and metabolic alterations, could play a key role in SCA7 and possibly other polyglutamine diseases.

  • 1060.
    Zadravec, Damir
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Wenner-Grens institut, Avdelningen för fysiologi.
    Tvrdik, Petr
    Guillou, Hervé
    Haslam, Richard
    Kobayashi, Tsutomu
    Stockholms universitet, Naturvetenskapliga fakulteten, Wenner-Grens institut.
    Napier, Johnathan A
    Capecchi, Mario R
    Jacobsson, Anders
    Stockholms universitet, Naturvetenskapliga fakulteten, Wenner-Grens institut, Avdelningen för fysiologi.
    ELOVL2 controls the level of n-6 28:5 and 30:5 fatty acids in testis: a prerequisite for male fertility and sperm maturation in mice2011Ingår i: Journal of Lipid Research, ISSN 0022-2275, E-ISSN 1539-7262, Vol. 52, nr 2, s. 245-255Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    ELOVL2 is a member of the mammalian microsomal ELOVL fatty acid enzyme family, involved in the elongation of very long-chain fatty acids including PUFAs required for various cellular functions in mammals. Here, we used ELOVL2-ablated (Elovl2(-/-)) mice to show that the PUFAs with 24-30 carbon atoms of the ω-6 family in testis are indispensable for normal sperm formation and fertility in male mice. The lack of Elovl2 was associated with a complete arrest of spermatogenesis, with seminiferous tubules displaying only spermatogonia and primary spermatocytes without further germinal cells. Furthermore, based on acyl-CoA profiling, heterozygous Elovl2(+/-) male mice exhibited haploinsufficiency, with reduced levels of C28:5 and C30:5n-6 PUFAs, which gave rise to impaired formation and function of haploid spermatides. These new insights reveal a novel mechanism involving ELOVL2-derived PUFAs in mammals and previously unrecognized roles for C28 and C30 n-6 PUFAs in male fertility. In accordance with the function suggested for ELOVL2, the Elovl2(-/-) mice show distorted levels of serum C20 and C22 PUFAs from both the n-3 and the n-6 series. However, dietary supplementation with C22:6n-3 could not restore male fertility to Elovl2(+/-) mice, suggesting that the changes in n-6 fatty acid composition seen in the testis of the Elovl2(+/-) mice, cannot be compensated by increased C22:6n-3 content.

  • 1061. Zaid, Ahmed
    Transcriptional regulation of human nuclear encoded mitochondrial genes2001Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Biogenesis of mammalian mitochondria requires the participation of both nuclear and mitochondrial genes. This thesis presents studies that characterize the common features of several nuclear encoded mitochondrial promoter genes and proposes the identity of factors regulating the transcription and responsible for coordinated, constitutive expression of diverse mammalian oxidative phosphorylation (OXPHOS) genes.

    As a model, we chose to study the promoters of four nuclear-encoded OXPHOS genes, all which are constitutively expressed, but, in addition, exhibit different responses to hormone and/or growth-activation. They also represent functionally different OXPHOS complexes. These promoters are from the human cytochrome c1, the mitochondrial transcription factor, mtTFA, the F1-ATPase b-subunit, and the adenine nucleotide translocator isoform 2 (ANT2) genes. The results presented in this thesis can be summarized as follows:

    The General transcription factor Sp1 can activate and repress different mammalian OXPHOS genes. Sp1 binding elements (GC boxes) are present in most, if not all, OXPHOS promoter, which suggest that Sp1 may have a general role in regulating the expression of nuclear encoded mitochondrial genes. (b) Sp1-mediated repression and activation from the ANT2 promoter require the D transactivation domain of Sp1 bound to the Cbox. (c) Sp1-mediated repression from the ANT2 promoter is not due to steric interference with the assembly of the transcription machinery. Sp1 bound to the Cbox, under certain conditions, activates the ANT2 promoter (d) Different Sp family members (Sp2, Sp3 and Sp4) differentially affect transcription of the OXPHOS promoters studied. (e) AP-2 enhances Sp1-dependent transactivation of the ANT2 promoter gene. (f) Sp3, but not Sp4, can repress the ANT2 promoter via the Cbox, suggesting that different members of the Sp family can have different roles when they bind to the ANT2 Cbox. (g) Thyroid hormone activates reporter gene expression driven from the ANT2 and cytochrome c1 promoters, which suggests that thyroid hormone induction of some OXPHOS genes is at the transcriptional level.

  • 1062. Zhang, Lifang
    et al.
    Carlberg, Inger
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Norling, Birgitta
    Deletion of Synechocystis sp PCC 6803 Leader Peptidase LepB1 Affects Photosynthetic Complexes and Respiration2013Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 12, nr 5, s. 1192-1203Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The cyanobacterium Synechocystis sp. PCC 6803 possesses two leader peptidases, LepB1 (SII0716) and LepB2 (SIr1377), responsible for the processing of signal peptide-containing proteins. Deletion of the gene for LepB1 results in an inability to grow photoautotrophically and an extreme light sensitivity. Here we show, using a combination of Blue Native/SDS-PAGE, Western blotting and iTRAQ analysis, that lack of LepB1 strongly affects the cell's ability to accumulate wild-type levels of both photosystem I (PSI) and cytochrome (Cyt) b(6)f complexes. The impaired assembly of PSI and Cyt b(6)f is considered to be caused by the no or slow processing of the integral subunits PsaF and Cyt f respectively. In particular, PsaF, one of the PSI subunits, was found incorporated into PSI in its unprocessed form, which could influence the assembly and/or stability of PSI. In contrast to these results, we found the amount of assembled photosystem II (PSII) unchanged, despite a slower processing of PsbO. Thus, imbalance in the ratios of PSI and Cyt b(6)f to photosystem II leads to an imbalanced photosynthetic electron flow up- and down-stream of the plastoquinone pool, resulting in the observed light sensitivity of the mutant. We conclude that LepB1 is the natural leader peptidase for PsaF, PsbO, and Cyt f. The maturation of PsbO and Cyt f can be partially performed by LepB2, whereas PsaF processing is completely dependent on LepB1. iTRAQ analysis also revealed a number of indirect effects accompanying the mutation, primarily a strong induction of the CydAB oxidase as well as a significant decrease in phycobiliproteins and chlorophyll/heme biosynthesis enzymes.

  • 1063.
    Zhang, Xuan
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Oglecka, Kamila
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Sandgren, Staffan
    Belting, Mattias
    Esbjorner, Elin K.
    Norden, Bengt
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Dual functions of the human antimicrobial peptide LL-37-Target membrane perturbation and host cell cargo delivery2010Ingår i: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1798, nr 12, s. 2201-2208Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The mechanisms behind target vs. host cell recognition of the human antimicrobial peptide LL-37 remain ill-defined. Here, we have investigated the membrane disruption capacity of LL-37 using large unilamellar vesicles (LUVs) composed of varying mixtures of POPC, POPG and cholesterol to mimic target and host membranes respectively. We show that LL-37 is unable to induce leakage of entrapped calcein from zwitterionic POPC LUVs, whereas leakage from LUVs partially composed of POPG is fast and efficient. In accordance with typical antimicrobial peptide behavior, cholesterol diminished LL-37 induced leakage. By using linear dichroism and flow oriented LUVs, we found that LL-37 orients with the axis of its induced alpha-helix parallel to the membrane surface in POPC:POPG (7:3) LUVs. In the same system, we also observed a time-dependent increase of the parallel alpha-helix LD signal on timescales corresponding to the leakage kinetics. The increased LD may be connected to a peptide translocation step, giving rise to mass balance across the membrane. This could end the leakage process before it is complete, similar to what we have observed. Confocal microscopy studies of eukaryotic cells show that LL-37 is able to mediate the cell delivery of non-covalently linked fluorescent oligonucleotides, in agreement with earlier studies on delivery of plasmid DNA (Sandgren et al., J. Biol. Chem. 279 (2004) 17951). These observations highlight the potential dual functions of LL-37 as an antimicrobial agent against bacterial target cells and a cell-penetrating peptide that can deliver nucleic acids into the host cells.

  • 1064.
    Zhang, Zhe
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Enhancing membrane and secretory protein production yields in Escherichia coli2020Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Proteins fulfill essential functions in living cells. To produce sufficient amounts of a protein is essential to study the structure and function of a protein, or to use it for medical purposes. Escherichia coli is a Gram-negative bacterium that is widely used for recombinant protein production. The aim of my PhD studies was to enhance membrane and secretory protein production yields using E. coli. The T7-based protein production system BL21(DE3)/pET was mainly used in my studies. BL21(DE3) contains a strong IPTG-inducible lacUV5 promoter governing the expression of the t7rnap gene encoding the T7RNAP on its chromosome. The target gene is under control of the T7 promoter on the pET plasmid. T7RNAP specifically recognizes the T7 promoter and transcribes the target gene more efficiently than the bacterial RNAP. Unfortunately, the biogenesis machinery for membrane and secretory proteins is usually saturated by the high protein production intensity when the BL21(DE3)/pET system is induced with IPTG, thereby negatively affecting protein production yields. In the first study, we found that when using the BL21(DE3)/pET system omitting the inducer IPTG improved membrane and secretory protein production yields. In previous studies, Lemo21(DE3) was developed to facilitate the production of membrane and secretory proteins. Lemo21(DE3) contains the pLemo plasmid in which the gene encoding the inhibitor of T7RNAP, T7 lysozyme, is under the control of the rhaBAD promoter. The activity of T7RNAP is regulated by synthesizing different levels of T7 lysozyme by adding different amounts of rhamnose. Thus, the production intensity can be modulated such that the biogenesis machinery of membrane and secretory proteins is not saturated upon IPTG induction. In the second study, we combined the key elements from both the pLemo and pET vectors to create the pReX (Regulated eXpression) plasmid to facilitate the use of helper plasmids encoding e.g., chaperones when it is necessary. In the third study, we used the rhaBAD promoter to direct the production of membrane and secretory proteins in a rhamnose metabolism and active uptake deficient strain. The protein production rate can be truly tuned in this setup. Therefore, the production of membrane and secretory proteins can be enhanced by using the right amount of rhamnose in the culture medium. BL21(DE3) contains the λDE3 prophage that carries the t7rnap gene under the control of the lacUV5 promoter. The λDE3 prophage is thought to be stably inserted into the chromosome, but the lytic cycle of the prophage can still be induced by the SOS response inducing antibiotic mitomycin C in the mitomycin C-based bacteriophage test. In the fourth study, we engineered BL21T7 by deleting in BL21(DE3) lysis related genes from the prophage. BL21T7 has similar recombinant protein production characteristics as its ancestor BL21(DE3).

  • 1065.
    Zhang, Zhe
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ampah-Korsah, Henry
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Karyolaimos, Alexandros
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    de Gier, Jan-Willem
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Construction and characterization of the bacteriophage testable BL21(DE3)-derivative BL21T7Manuskript (preprint) (Övrigt vetenskapligt)
  • 1066.
    Zhou, Shu
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Pettersson, Pontus
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Björck, Markus L.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Dawitz, Hannah
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Brzezinski, Peter
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ädelroth, Pia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    NMR structural analysis of yeast Cox13 reveals its C-terminus in interaction with ATPManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    The organization of mitochondrial respiratory chain complexes into supercomplexes is vital to cellular activities. In the yeast Saccharomyces cerevisiae, Cox13 is a conserved peripheral subunit of complex IV (cytochrome c oxidase, CytcO) involved in the assembly of monomeric complex IV into supercomplexes. Here we report the solution NMR structure of a Cox13 dimer in detergent micelles. Each Cox13 monomer has three short flexible helices (SH), corresponding to the disordered regions in its homologous X-ray structure, and the dimer formation is mainly induced by the hydrophobic interaction between the sole transmembrane (TM) helix of each monomer. Furthermore, analysis of chemical shift changes upon addition of ATP reveal positions that are able to bind ATP at the conserved sites of the C-terminus with considerable conformational flexibility. From functional analysis of purified CytcO, we conclude that this ATP interaction is inhibitory of catalytic activity. Our results show the structure of an important subunit of yeast CytcO and provide structure-based insight into its ATP interaction.

  • 1067.
    Zhou, Tuping
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för material- och miljökemi (MMK).
    Shu, Nanjiang
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för material- och miljökemi (MMK).
    Hovmöller, Sven
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för material- och miljökemi (MMK).
    A Novel Method for Accurate One-dimensional Protein Structure Prediction Based on Fragment Matching2010Ingår i: Bioinformatics, ISSN 1367-4803, E-ISSN 1367-4811, Vol. 26, nr 4, s. 470-477Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Motivation: The precise prediction of one-dimensional (1D) protein structure as represented by the protein secondary structure and 1D string of discrete state of dihedral angles (i.e. Shape Strings) is a prerequisite for the successful prediction of three-dimensional (3D) structure as well as protein-protein interaction. We have developed a novel 1D structure prediction method, called Frag1D, based on a straightforward fragment matching algorithm and demonstrated its success in the prediction of  three sets of 1D structural alphabets, i.e. the classical three-state secondary structure, three-state Shape Strings and eight-state Shape Strings.

    Results: By exploiting the vast protein sequence and protein structure data available, we have brought secondary structure prediction closer to the expected theoretical limit. When tested by a leave-one-out cross validation on a non-redundant set of PDB cutting at 30% sequence identity containing 5860 protein chains, the overall per-residue accuracy for secondary structure prediction, i.e. Q3 is 82.9%. The overall per-residue accuracy for three-state and eight-state Shape Strings are 85.1% and 71.5% respectively. We have also benchmarked our program with the latest version of PSIPRED for secondary structure prediction and our program predicted 0.3% better in Q3 when tested on 2241 chains with the same training set. For Shape Strings, we compared our method with a recently published method with the same dataset and definition as used by that method. Our program predicted at 2.2% better in accuracy for three-state Shape Strings. By quantitatively investigating the effect of data base size on 1D structure prediction we show that the accuracy increases by about 1% with every doubling of the database size.

  • 1068. Zhu, Haining
    et al.
    Zhao, Jun
    Zhu, Beibei
    Collazo, Joanne
    Gal, Jozsef
    Shi, Ping
    Liu, Li
    Ström, Anna-Lena
    University of Kentucky, USA.
    Lu, Xiaoning
    McCann, Richard O.
    Toborek, Michal
    Kyprianou, Natasha
    EMMPRIN regulates cytoskeleton reorganization and cell adhesion in prostate cancer2011Ingår i: The Prostate, ISSN 0270-4137, E-ISSN 1097-0045, Vol. 72, nr 1, s. 72-81Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    BACKGROUND: Proteins on cell surface play important roles during cancer progression and metastasis via their ability to mediate cell-to-cell interactions and navigate the communication between cells and the microenvironment. METHODS: In this study a targeted proteomic analysis was conducted to identify the differential expression of cell surface proteins in human benign (BPH-1) versus malignant (LNCaP and PC-3) prostate epithelial cells. We identified EMMPRIN (extracellular matrix metalloproteinase inducer) as a key candidate and shRNA functional approaches were subsequently applied to determine the role of EMMPRIN in prostate cancer cell adhesion, migration, invasion as well as cytoskeleton organization. RESULTS: EMMPRIN was found to be highly expressed on the surface of prostate cancer cells compared to BPH-1 cells, consistent with a correlation between elevated EMMPRIN and metastasis found in other tumors. No significant changes in cell proliferation, cell cycle progression, or apoptosis were detected in EMMPRIN knockdown cells compared to the scramble controls. Furthermore, EMMPRIN silencing markedly decreased the ability of PC-3 cells to form filopodia, a critical feature of invasive behavior, while it increased expression of cell-cell adhesion and gap junction proteins. CONCLUSIONS: Our results suggest that EMMPRIN regulates cell adhesion, invasion, and cytoskeleton reorganization in prostate cancer cells. This study identifies a new function for EMMPRIN as a contributor to prostate cancer cell-cell communication and cytoskeleton changes towards metastatic spread, and suggests its potential value as a marker of prostate cancer progression to metastasis.

  • 1069. Zuber, Bartek
    et al.
    Rudstrom, Karin
    Ehrnfelt, Cecilia
    Ahlborg, Niklas
    Stockholms universitet, Naturvetenskapliga fakulteten, Wenner-Grens institut, Avdelningen för immunologi. Mabtech, Sweden.
    Epitope Mapping of Neutralizing Monoclonal Antibodies to Human Interferon-gamma Using Human-Bovine Interferon-gamma Chimeras2016Ingår i: Journal of Interferon and Cytokine Research, ISSN 1079-9907, E-ISSN 1557-7465, Vol. 36, nr 9, s. 542-551Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Our aim was to identify conformational epitopes, recognized by monoclonal antibodies (mAbs) made against human (h) interferon (IFN)-. Based on the mAbs' (n=12) ability to simultaneously bind hIFN- in ELISA, 2 epitope clusters with 5mAbs in each were defined; 2mAbs recognized unique epitopes. Utilizing the mAbs' lack of reactivity with bovine (b) IFN-, epitopes were identified using 7h/bIFN- chimeras where the helical regions (A-F) or the C terminus were substituted with bIFN- residues. Chimeras had a N-terminal peptide tag enabling the analysis of mAb recognition of chimeras in ELISA. The 2mAb clusters mapped to region A and E, respectively; the epitopes of several mAbs also involved additional regions. MAbs in cluster A neutralized, to various degrees, IFN--mediated activation of human cells, in line with the involvement of region A in the IFN- receptor interaction. MAbs mapping to region E displayed a stronger neutralizing capacity although this region has not been directly implicated in the receptor interaction. The results corroborate earlier studies and provide a detailed picture of the link between the epitope specificity and neutralizing capacity of mAbs. They further demonstrate the general use of peptide-tagged chimeric proteins as a powerful and straightforward method for efficient mapping of conformational epitopes.

  • 1070.
    Öhrström, Maria
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Popović-Bijelić, Ana
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Luo, Jinghui
    Stenmark, Pål
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Högbom, Martin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Inhibition of chlamydial class Ic ribonucleotide reductase by C-terminal peptides from protein R22011Ingår i: Journal of Peptide Science, ISSN 1075-2617, E-ISSN 1099-1387, Vol. 17, nr 11, s. 756-762Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Chlamydia trachomatis ribonucleotide reductase (RNR) is a class Ic RNR. It has two homodimeric subunits: proteins R1 and R2. Class Ic protein R2 in its most active form has a manganese-iron metal cofactor, which functions in catalysis like the tyrosyl radical in classical class Ia and Ib RNRs. Oligopeptides with the same sequence as the C-terminus of C. trachomatis protein R2 inhibit the catalytic activity of C. trachomatis RNR, showing that the class Ic enzyme shares a similar highly specific inhibition mechanism with the previously studied radical-containing class Ia and Ib RNRs. The results indicate that the catalytic mechanism of this class of RNRs with a manganese-iron cofactor is similar to that of the tyrosyl-radical-containing RNRs, involving reversible long-range radical transfer between proteins R1 and R2. The competitive binding of the inhibitory R2-derived oligopeptide blocks the transfer pathway. We have constructed three-dimensional structure models of C. trachomatis protein R1, based on homologous R1 crystal structures, and used them to discuss possible binding modes of the peptide to protein R1. Typical half maximal inhibitory concentration values for C. trachomatis RNR are about 200 µ m for a 20-mer peptide, indicating a less efficient inhibition compared with those for an equally long peptide in the Escherichia coli class Ia RNR. A possible explanation is that the C. trachomatis R1/R2 complex has other important interactions, in addition to the binding mediated by the R1 interaction with the C-terminus of protein R2.

  • 1071.
    Öjemalm, Karin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Membrane protein topogenesis2015Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    The membranes of cells are highly complex and heterogeneous structures that fulfill multiple vital tasks. They form thin barriers that seal out the environment, thus defining the cell’s boundaries. They mediate the selective exchange of information and substances between the inside and outside of cells, thus making cellular life and survival possible and allowing fast adaptation to changing conditions. Not least importantly, they harbor key components of many essential processes such as the photosynthesis and respiration. Membranes are composed of a largely apolar lipid matrix densely punctuated with a large number of different proteins. These so-called membrane proteins usually span the lipid matrix and protrude out into the space on either side of the membrane.

    Over millions of years of evolution, cells have developed an incredible machinery to facilitate the insertion of membrane proteins into the membrane. Our understanding of these machines and the insertion processes they mediate has reached a point where we have a very good picture of membrane protein biogenesis in various types of cells. However, more data still needs to be collected to completely comprehend the complex molecular mechanisms and the physical chemistry that underlies the different insertion processes.

    The work presented in this thesis contributes to that understanding. More precisely, we have studied how weakly hydrophobic transmembrane elements of membrane proteins, which cannot spontaneously enter the largely apolar membrane matrices, are efficiently incorporated. Indeed, such elements are quite common in membrane proteins and our work has lead to the formulation of a novel mechanism for how they can be integrated into biological membranes.

    We have also added to the understanding of the physical chemistry that underlies the membrane insertion process by systematically introducing non-proteinogenic amino acids into a membrane-spanning segment of a membrane protein and studying its membrane insertion capability.

  • 1072.
    Öjemalm, Karin
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Calado Botelho, Salomé
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Stüdle, Chiara
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    von Heijne, Gunnar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Quantitative Analysis of SecYEG-Mediated Insertion of Transmembrane alpha-Helices into the Bacterial Inner Membrane2013Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 425, nr 15, s. 2813-2822Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Most integral membrane proteins, both in prokaryotic and eukaryotic cells, are co-translationally inserted into the membrane via Sec-type translocons: the SecYEG complex in prokaryotes and the Sec61 complex in eukaryotes. The contributions of individual amino acids to the overall free energy of membrane insertion of single transmembrane alpha-helices have been measured for Sec61-mediated insertion into the endoplasmic reticulum (ER) membrane (Nature 450:1026-1030) but have not been systematically determined for SecYEG-mediated insertion into the bacterial inner membrane. We now report such measurements, carried out in Escherichia coli. Overall, there is a good correlation between the results found for the mammalian ER and the E. coli inner membrane, but the hydrophobicity threshold for SecYEG-mediated insertion is distinctly lower than that for Sec61-mediated insertion.

  • 1073.
    Öjemalm, Karin
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Higuchi, Takashi
    Jiang, Yang
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Nilsson, IngMarie
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    White, Stephen H.
    Suga, Hiroaki
    von Heijne, Gunnar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Apolar surface area determines the efficiency of translocon-mediated membrane-protein integration into the endoplasmic reticulum2011Ingår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 108, nr 31, s. E359-E364Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Integral membrane proteins are integrated cotranslationally into the membrane of the endoplasmic reticulum in a process mediated by the Sec61 translocon. Transmembrane α-helices in a translocating polypeptide chain gain access to the surrounding membrane through a lateral gate in the wall of the translocon channel [van den Berg B, et al. (2004) Nature427:36–44; Zimmer J, et al. (2008) Nature455:936–943; Egea PF, Stroud RM (2010)Proc Natl Acad Sci USA 107:17182–17187]. To clarify the nature of the membrane-integration process, we have measured the insertion efficiency into the endoplasmic reticulum membrane of model hydrophobic segments containing nonproteinogenic aliphatic and aromatic amino acids. We find that an amino acid’s contribution to the apparent free energy of membrane-insertion is directly proportional to the nonpolar accessible surface area of its side chain, as expected for thermodynamic partitioning between aqueous and nonpolar phases. But unlike bulk-phase partitioning, characterized by a nonpolar solvation parameter of 23 cal∕ðmol · Å2Þ, the solvation parameter for transfer from translocon to bilayer is 6 –10 cal∕ðmol · Å2Þ, pointing to important differences between translocon-guided partitioning and simple water-to-membrane partitioning. Our results provide compelling evidence for a termodynamic partitioning model and insights into the physical properties of the translocon.

  • 1074.
    Öjemyr, Linda
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Sandén, Tor
    Widengren, Jerker
    Brzezinski, Peter
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lateral proton transfer between the membrane and a membrane protein.2009Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, nr 10, s. 2173-9Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Proton transport across biological membranes is a key step of the energy conservation machinery in living organisms, and it has been proposed that the membrane itself plays an important role in this process. In the present study we have investigated the effect of incorporation of a proton transporter, cytochrome c oxidase, into a membrane on the protonation kinetics of a fluorescent pH-sensitive probe attached at the surface of the protein. The results show that proton transfer to the probe was slightly accelerated upon attachment at the protein surface (approximately 7 x 1010 s(-1) M(-1), compared to the expected value of (1-2) x 10(10) s(-1) M(-1)), which is presumably due to the presence of acidic/His groups in the vicinity. Upon incorporation of the protein into small unilamellar phospholipid vesicles the rate increased by more than a factor of 400 to approximately 3 x 10(13) s(-1) M(-1), which indicates that the protein-attached probe is in rapid protonic contact with the membrane surface. The results indicate that the membrane acts to accelerate proton uptake by the membrane-bound proton transporter.

  • 1075.
    Östbye, Henrik
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    da Silva, Diogo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Revol, Rebecca
    Nordholm, Johan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Assembly co-cooperativity between the influenza 1 NA stalk and 2 transmembrane domain defines the insertion deletion boundaryManuskript (preprint) (Övrigt vetenskapligt)
19202122 1051 - 1075 av 1075
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