| 1. |
- Blockhuys, Stephanie, 1983, et al.
(författare)
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Defining the human copper proteome and analysis of its expression variation in cancers.
- 2017
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Ingår i: Metallomics. - : Oxford University Press (OUP). - 1756-5901 .- 1756-591X. ; 9:2, s. 112-123
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Tidskriftsartikel (refereegranskat)abstract
- Copper (Cu) is essential for living organisms, and acts as a cofactor in many metabolic enzymes. To avoid the toxicity of free Cu, organisms have specific transport systems that 'chaperone' the metal to targets. Cancer progression is associated with increased cellular Cu concentrations, whereby proliferative immortality, angiogenesis and metastasis are cancer hallmarks with defined requirements for Cu. The aim of this study is to gather all known Cu-binding proteins and reveal their putative involvement in cancers using the available database resources of RNA transcript levels. Using the database along with manual curation, we identified a total of 54 Cu-binding proteins (named the human Cu proteome). Next, we retrieved RNA expression levels in cancer versus normal tissues from the TCGA database for the human Cu proteome in 18 cancer types, and noted an intricate pattern of up- and downregulation of the genes in different cancers. Hierarchical clustering in combination with bioinformatics and functional genomics analyses allowed for the prediction of cancer-related Cu-binding proteins; these were specifically inspected for the breast cancer data. Finally, for the Cu chaperone ATOX1, which is the only Cu-binding protein proposed to have transcription factor activities, we validated its predicted over-expression in patient breast cancer tissue at the protein level. This collection of Cu-binding proteins, with RNA expression patterns in different cancers, will serve as an excellent resource for mechanistic-molecular studies of Cu-dependent processes in cancer.
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| 2. |
- Ulmefors, Hanna, et al.
(författare)
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Formation of Supported Lipid Bilayers Derived from Vesicles of Various Compositional Complexity on Conducting Polymer/Silica Substrates
- 2021
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Ingår i: Langmuir. - : American Chemical Society (ACS). - 0743-7463 .- 1520-5827. ; 37:18, s. 5494-5505
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Tidskriftsartikel (refereegranskat)abstract
- Supported lipid bilayers (SLBs) serve important roles as minimalistic models of cellular membranes in multiple diagnostic and pharmaceutical applications as well as in the strive to gain fundamental insights about their complex biological function. To further expand the utility of SLBs, there is a need to go beyond simple lipid compositions to thereby better mimic the complexity of native cell membranes, while simultaneously retaining their compatibility with a versatile range of analytical platforms. To meet this demand, we have in this work explored SLB formation on PEDOT:PSS/silica nanoparticle composite films and mesoporous silica films, both capable of transporting ions to an underlying conducting PEDOT:PSS film. The SLB formation process was evaluated by using the quartz crystal microbalance with dissipation (QCM-D) monitoring, total internal reflection fluorescence (TIRF) microscopy, and fluorescence recovery after photobleaching (FRAP) for membranes made of pure synthetic lipids with or without the reconstituted membrane protein β-secretase 1 (BACE1) as well as cell-derived native lipid vesicles containing overexpressed BACE1. The mesoporous silica thin film was superior to the PEDOT:PSS/silica nanoparticle composite, providing successful formation of bilayers with high lateral mobility and low defect density even for the most complex native cell membranes.
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| 3. |
- Conti, Luca, et al.
(författare)
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Reciprocal voltage sensor-to-pore coupling leads to potassium channel C-type inactivation
- 2016
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Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- Voltage-gated potassium channels open at depolarized membrane voltages. A prolonged depolarization causes a rearrangement of the selectivity filter which terminates the conduction of ions - a process called slow or C-type inactivation. How structural rearrangements in the voltage-sensor domain (VSD) cause alteration in the selectivity filter, and vice versa, are not fully understood. We show that pulling the pore domain of the Shaker potassium channel towards the VSD by a Cd2+ bridge accelerates C-type inactivation. Molecular dynamics simulations show that such pulling widens the selectivity filter and disrupts the K+ coordination, a hallmark for C-type inactivation. An engineered Cd2+ bridge within the VSD also affect C-type inactivation. Conversely, a pore domain mutation affects VSD gating-charge movement. Finally, C-type inactivation is caused by the concerted action of distant amino acid residues in the pore domain. All together, these data suggest a reciprocal communication between the pore domain and the VSD in the extracellular portion of the channel.
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| 4. |
- Malmberg, Per, 1974, et al.
(författare)
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Formation of hydroxyapatite on titanium implants in vivo precedes bone-formation during healing
- 2017
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Ingår i: Biointerphases. - : American Vacuum Society. - 1934-8630 .- 1559-4106. ; 12:4
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Tidskriftsartikel (refereegranskat)abstract
- The bone material interface has been an area of intense study over many decades, where studies of the healing process ranging from simple mineral deposition in vitro to actual healing in vivo have given important clues to the importance of calcium minerals in the bone/implant interface. Here, the authors use a combination of in vitro cell culture methods and in vivo implantation to study how the role of the spontaneously formed hydroxyapatite layer on Ti-implants for the in vivo-healing into the bone tissue of rat tibia. Initial experiments were made in reduced systems by incubation of TiO2 in cell culture medium and analysis by time of flight secondary ion mass spectrometry (ToF-SIMS) and energy-dispersive x-ray spectroscopy followed by subsequent exposure of human embryological stem cells analyzed by von Kossa staining and environmental scanning electron microsopy. In vivo studies of the bone-material interface was analyzed by ToF-SIMS depth profiling using both C-60(+) ions as well as a gas cluster ion source beam, Ar-1500(+) as sputter source. The low ion yield of the Ar-1500(+) for inorganics allowed the inorganic/organic interface of the implant to be studied avoiding the erosion of the inorganic materials caused by the conventional C-60(+) beam. (C) 2017 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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| 5. |
- Nilsson, Michelle, et al.
(författare)
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Voltage-dependent G-protein regulation of CaV2.2 (N-type) channels
- 2024
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Ingår i: Science Advances. - 2375-2548. ; 10:37
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Tidskriftsartikel (refereegranskat)abstract
- How G proteins inhibit N-type, voltage-gated, calcium-selective channels (CaV2.2) during presynaptic inhibition is a decades-old question. G proteins Gβγ bind to intracellular CaV2.2 regions, but the inhibition is voltage dependent. Using the hybrid electrophysiological and optical approach voltage-clamp fluorometry, we show that Gβγ acts by selectively inhibiting a subset of the four different CaV2.2 voltage-sensor domains (VSDs I to IV). During regular “willing” gating, VSD-I and -IV activations resemble pore opening, VSD III activation is hyperpolarized, and VSD II appears unresponsive to depolarization. In the presence of Gβγ, CaV2.2 gating is “reluctant”: pore opening and VSD I activation are strongly and proportionally inhibited, VSD IV is modestly inhibited, while VSD III is not. We propose that Gβγ inhibition of VSDs I and IV underlies reluctant CaV2.2 gating and subsequent presynaptic inhibition.
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| 6. |
- Aronsson, Christopher
(författare)
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Tunable and modular assembly of polypeptides and polypeptide-hybrid biomaterials
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Biomaterials are materials that are specifically designed to be in contact with biological systems and have for a long time been used in medicine. Examples of biomaterials range from sophisticated prostheses used for replacing outworn body parts to ordinary contact lenses. Currently it is possible to create biomaterials that can e.g. specifically interact with cells or respond to certain stimuli. Peptides, the shorter version of proteins, are excellent molecules for fabrication of such biomaterials. By following and developing design rules it is possible to obtain peptides that can self-assemble into well-defined nanostructures and biomaterials.The aim of this thesis is to create ”smart” and tunable biomaterials by molecular self-assembly using dimerizing –helical polypeptides. Two different, but structurally related, polypeptide-systems have been used in this thesis. The EKIV-polypeptide system was developed in this thesis and consists of four 28-residue polypeptides that can be mixed-and-matched to self-assemble into four different coiled coil heterodimers. The dissociation constant of the different heterodimers range from μM to < nM. Due to the large difference in affinities, the polypeptides are prone to thermodynamic social self-sorting. The JR-polypeptide system, on the other hand, consists of several 42-residue de novo designed helix-loop-helix polypeptides that can dimerize into four-helix bundles. In this work, primarily the glutamic acid-rich polypeptide JR2E has been explored as a component in supramolecular materials. Dimerization was induced by exposing the polypeptide to either Zn2+, acidic conditions or the complementary polypeptide JR2K.By conjugating JR2E to hyaluronic acid and the EKIV-polypeptides to star-shaped poly(ethylene glycol), respectively, highly tunable hydrogels that can be self-assembled in a modular fashion have been created. In addition, self-assembly of spherical superstructures has been investigated and were obtained by linking two thiol-modified JR2E polypeptides via a disulfide bridge in the loop region. ŒThe thesis also demonstrates that the polypeptides and the polypeptide-hybrids can be used for encapsulation and release of molecules and nanoparticles. In addition, some of the hydrogels have been explored for 3D cell culture. By using supramolecular interactions combined with bio-orthogonal covalent crosslinking reactions, hydrogels were obtained that enabled facile encapsulation of cells that retained high viability.The results of the work presented in this thesis show that dimerizing α–helical polypeptides can be used to create modular biomaterials with properties that can be tuned by specific molecular interactions. The modularity and the tunable properties of these smart biomaterials are conceptually very interesting andmake them useful in many emerging biomedical applications, such as 3D cell culture, cell therapy, and drug delivery.
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| 7. |
- Carlberg, Inger, et al.
(författare)
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A novel plant protein undergoing light-induced phosphorylation and release from the photosynthetic thylakoid membranes
- 2003
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 100:2, s. 757-62
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Tidskriftsartikel (refereegranskat)abstract
- The characteristics of a phosphoprotein with a relative electrophoretic mobility of 12 kDa have been unknown during two decades of studies on redox-dependent protein phosphorylation in plant photosynthetic membranes. Digestion of this protein from spinach thylakoid membranes with trypsin and subsequent tandem nanospray-quadrupole-time-of-flight mass spectrometry of the peptides revealed a protein sequence that did not correspond to any previously known protein. Sequencing of the corresponding cDNA uncovered a gene for a precursor protein with a transit peptide followed by a strongly basic mature protein with a molecular mass of 8,640 Da. Genes encoding homologous proteins were found on chromosome 3 of Arabidopsis and rice as well as in ESTs from 20 different plant species, but not from any other organisms. The protein can be released from the membrane with high salt and is also partially released in response to light-induced phosphorylation of thylakoids, in contrast to all other known thylakoid phosphoproteins, which are integral to the membrane. On the basis of its properties, this plant-specific protein is named thylakoid soluble phosphoprotein of 9 kDa (TSP9). Mass spectrometric analyses revealed the existence of non-, mono-, di-, and triphosphorylated forms of TSP9 and phosphorylation of three distinct threonine residues in the central part of the protein. The phosphorylation and release of TSP9 from the photosynthetic membrane on illumination favor participation of this basic protein in cell signaling and regulation of plant gene expression in response to changing light conditions.
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| 8. |
- Silverå Ejneby, Malin, 1987-
(författare)
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Site and Mechanism of Action of Resin Acids on Voltage-Gated Ion Channels
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Voltage-gated ion channels are pore-forming membrane proteins that open or close their gates when the voltage across the membrane is changed. They underlie the electrical activity that enables the heart to pump blood and the brain to receive and send signals. Changes in expression, distribution, and functional properties of voltage-gated ion channels can lead to diseases, such as epilepsy, cardiac arrhythmia, and pain-related disorders. Drugs that modulate the function of voltage-gated ion channels control these diseases in some patients, but the existing drugs do not adequately help all patients, and some also have severe side effects.Resin acids are common components of pine resins, with a hydrophobic three-ringed motif and a negatively charged carboxyl group. They open big-conductance Ca2+-activated K+ (BK) channels and voltage-gated potassium (KV) channels. We aimed to characterize the binding site and mechanism of action of resin acids on a KV channel and explore the effect of a resin acid by modifying the position and valence of charge of the carboxyl group. We tested the effect on several voltage-gated ion channels, including two KV channels expressed in Xenopus laevis oocytes and several voltage-gated ion channels expressed in cardiomyocytes. For this endeavour different electrophysiological techniques, ion channels, and cell types were used together with chemical synthesis of about 140 resin-acid derivatives, mathematical models, and computer simulations.We found that resin acids bind between the lipid bilayer and the Shaker KV channel, in the cleft between transmembrane segment S3 and S4, on the extracellular side of the voltage-sensor domain. This is a fundamentally new interaction site for small-molecule compounds that otherwise usually bind to ion channels in pockets surrounded by water. We also showed that the resin acids open the Shaker KV channel via an electrostatic mechanism, exerted on the positively charged voltage sensor S4. The effect of a resin acid increased when the negatively charged carboxyl group (the effector) and the hydrophobic three-ringed motif (anchor in lipid bilayer) were separated by three atoms: longer stalks decreased the effect. The length rule, in combination with modifications of the anchor, was used to design new resin-acid derivatives that open the human M-type (Kv7.2/7.3) channel. A naturally occurring resin acid also reduced the excitability of cardiomyocytes by affecting the voltage-dependence of several voltage-gated ion channels. The major finding was that the resin acid inactivated sodium and calcium channels, while it activated KV channels at more negative membrane voltages. Computer simulations confirmed that the combined effect on different ion channels reduced the excitability of a cardiomyocyte. Finally, the resin acid reversed induced arrhythmic firing of the cardiomyocytes.In conclusion, resin acids are potential drug candidates for diseases such as epilepsy and cardiac arrhythmia: knowing the binding site and mechanism of action can help to fine tune the resin acid to increase the effect, as well as the selectivity.
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| 9. |
- Skyttner, Camilla, 1985-
(författare)
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Peptide-Liposome Model Systems for Triggered Release
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Liposomes are widely used in drug delivery to improve drug efficacy and to reduce side effects. For liposome-encapsulated drugs to become bioavailable and provide a therapeutic effect they must be released, which typically is a slow process that primarily relies on passive diffusion, liposome rupture or endocytotic uptake. Achieving drug concentrations within the therapeutic window can thus be challenging, resulting in poor efficacy and higher risks drug resistance. Finding means to modulate lipid membrane integrity and to trigger rapid and efficient release of liposomal cargo is thus critical to improve current and future liposomal drug delivery systems. The possibilities to tailor lipid composition and surface functionalization is vital for drug delivery applications but also make liposomes attractive model systems for studies of membrane active biomolecules.The overall aim of this thesis work has been to develop new strategies for triggering and controlling changes in lipid membrane integrity and to study the interactions of membrane active peptides with model lipid membranes using both de novo designed and biologically derived synthetic amphipathic cationic peptides. Two different sets of designed peptides have been explored that can fold and heterodimerize into a coiled coil and helix-loop-helix fourhelix bundle, respectively. Conjugation of the cationic lysine rich peptides to liposomes triggered a rapid and concentration dependent release. The additions of their corresponding glutamic acid-rich complementary peptides inhibited the release of liposomal cargo. Possibilities to reduce the inhibitory effect by both proteolytic digestion of the inhibitory peptide and by means of heterodimer exchange have been investigated. Moreover, the effects of peptide size and composition and ability to fold have been studied in order to elucidate the factors that influence the membrane permeabilizing effects of the peptides.In addition, the membrane activity of a the two-peptide bacteriocin PLNC8α and PLNC8β has been explored using liposomes as a model system. PLNC8αβ are expressed by Lactobacillus plantarum and were shown to display pronounced membrane-partition folding coupling, leading to rapid release of liposome encapsulated carboxyfluorescein. PLNC8αβ also kill and suppressed growth of the gram-negative bacteria Porphyromonas gingivalis by efficiently damaging the bacterial membrane.Although membrane active peptides are highly efficient in perturbing lipid membrane integrity, possibilities to trigger release using external stimuli are also of large interest for therapeutic applications. Light-induced heating of liposome encapsulated gold nanoparticles (AuNPs) has been shown by others as a potential strategy to trigger drug release. To facilitate fabrication of thermoplasmonic liposome systems we developed a simple method for synthesis of small AuNPs inside liposomes, using the liposomes as nanoscale reaction vessels.The work presented in this thesis provides new knowledge and techniques for future development of liposome-based drug delivery systems, peptide-based therapeutics and increase our understanding of peptide-lipid interactions.
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| 10. |
- Turkina, Maria V, et al.
(författare)
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The transit peptide of CP29 thylakoid protein in Chlamydomonas reinhardtii is not removed but undergoes acetylation and phosphorylation
- 2004
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Ingår i: FEBS Letters. - Amsterdam : Elsevier. - 0014-5793 .- 1873-3468. ; 564:1-2, s. 104-108
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Tidskriftsartikel (refereegranskat)abstract
- The surface-exposed peptides were cleaved by trypsin from the photosynthetic thylakoid membranes isolated from the green alga Chlamydomonas reinhardtii. Two phosphorylated peptides, enriched from the peptide mixture and sequenced by nanospray quadrupole time-of-flight mass spectrometry, revealed overlapping sequences corresponding to the N-terminus of a nuclear-encoded chlorophyll a/b-binding protein CP29. In contrast to all known nuclear-encoded thylakoid proteins, the transit peptide in the mature algal CP29 was not removed but processed by methionine excision, N-terminal acetylation and phosphorylation on threonine 6. The importance of this phosphorylation site is proposed as the reason of the unique transit peptide retention.
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