Mass-spectrometric analysis of glycerophospholipid metabolism

This thesis consists of five parts. In the first part, the first automated method for quantitative analysis of lipid compositions of cells and tissues by liquid chromatography-mass spectrometry was developed. In the second part, this method was applied to investigate brain lipid compositions of patients with progressive epilepsy with mental retardation (EPMR), caused by mutations in the CLN8 gene. We were able to show major progressive alterations in brain lipid profiles of EPMR patients which may contribute to disease pathogenesis in those patients. In the third part, a novel approach to investigate the metabolism of single glycerophospholipid molecular species in living cells was developed. This approach was applied to study mechanisms of acyl chain remodeling, i.e. the exchange of fatty acyl residues, of aminophospholipids in BHK and HeLa cells. In the fourth part a novel mass-spectrometric approach was developed to investigate the substrate specificity of phospholipases and was utilized to elucidate the specificities of secretory A-type phospholipases in unprecedented detail. We showed that the specificity of those phospholipases depended mainly on the propensity of the substrates to efflux from the membrane and interactions between the substrate and the enzyme catalytic site are secondary. In the fifth part of this thesis, mechanisms of mammalian glycerophospholipid homeostasis were reviewed and novel theoretical considerations presented.Tämä väitöskirjatyö koostuu viidestä osasta. Ensimmäisessä osatyössä kehitimme ensimmäisen automatisoidun nestekromatografia-massaspektrometriaan perustuvan menetelmän solujen ja kudosten lipidikoostumusten määrittämiseen. Toisessa osatyössä hyödynsimme em. menetelmää tarkastellaksemme lipidikoostumuksia ns. pohjoista epilepsiaa (EPMR) sairastavien potilaiden aivoista. Osoitimme, että potilasaivojen lipidikoostumuksessa tapahtuu progressiivisia muutoksia, jotka saattavat vaikuttaa taudin patogeneesiin. Kolmannessa osassa kehitimme uuden lähestymistavan yksittäisten fosfolipidimolekyylien aineenvaihdunnan tutkimiseen elävissä soluissa. Tätä menetelmää hyödyntäen tutkimme fosfolipidien rasvahappoketjujen muokkauksen mekanismeja BHK ja HeLa soluissa. Neljännessä osatyössä kehitettiin massaspektrometriaan perustuva menetelmä fosfolipaasien substraattispesifisyyden tutkimiseen. Em. menetelmää hyödyntäen määritimme kolmen ns. sekretorisen A-tyypin fosfolipaasin substraattispesifisyyden ennennäkemättömällä tarkkuudella. Osoitimme, että näiden fosfolipaasien substraattispesifisyyden määrää pääosin substraattien taipumus irtautua kalvosta ja substraatin ja katalyyttisen keskuksen väliset vuorovaikutukset ovat toissijaisia. Väitöskirjatyön viides osa on katsaus-tyyppinen tarkastelu glyserofosfolipidihomeostaasin mekanismeista, jossa esitetään myös uusia teoreettisia pohdintoja

Hypothesis : Chemical activity regulates and coordinates the processes maintaining glycerophospholipid homeostasis in mammalian cells

Mammalian cells maintain the complex glycerophospholipid (GPL) class compositions of their various membranes within close limits because this is essential to their well-being or viability. Surprisingly, however, it is still not understood how those compositions are maintained except that GPL synthesis and degradation are closely coordinated. Here, we hypothesize that abrupt changes in the chemical activity of the individual GPL classes coordinate synthesis and degradation as well other the homeostatic processes. We have previously proposed that only a limited number of “allowed” or “optimal” GPL class compositions exist in cellular membranes because those compositions are energetically more favorable than others, that is, they represent local free energy minima (Somerharju et al 2009, Biochim. Biophys. Acta 1788, 12-23). This model, however, could not satisfactorily explain how the “optimal” compositions are sensed by the key homeostatic enzymes, that is, rate-limiting synthetizing enzymes and homeostatic phospholipases. We now hypothesize that when the mole fraction of a GPL class exceeds an optimal value, its chemical activity abruptly increases which (a) increases its propensity to efflux from the membrane thus making it susceptible for hydrolysis by homeostatic phospholipases; (b) increases its potency to inhibit its own biosynthesis via a feedback mechanism; (c) enhances its conversion to another glycerophospholipid class via a novel process termed “head group remodeling” or (d) enhances its translocation to other subcellular membranes. In summary, abrupt change in the chemical activity of the individual GPL classes is proposed to regulate and coordinate those four processes maintaining GPL class homeostasis in mammalian cells.Peer reviewe

The PNPLA-family phospholipases involved in glycerophospholipid homeostasis of HeLa cells

Mammalian cells maintain the glycerophospholipid (GPL) compositions of their membranes nearly constant. To achieve this, GPL synthesis and degradation must be coordinated. There is strong evidence that A-type phospholipases (PLAs) are key players in homeostatic degradation of GPLs, but the identities of the PLAs involved have not been established. However, some members of the Patatin-like phospholipase domain-containing proteins (PNPLAs) have been implicated. Accordingly, we knocked down all the PNPLAs significantly expressed in human HeLa cells using RNA interference and then determined whether the turnover of the major glycerophospholipids is affected by using mass spectrometry and metabolic labeling with stable isotope labeled precursors. Knockdown of PNPLA9, PNPLA6 or PNPLA4 significantly (30-50%) reduced the turnover of phosphatidylcholine, ethanolamine and-serine. In a notable contrast, turnover of phosphatidylinositol was not significantly affected by the knockdown of any PNPLA. Depletion of PNPLA9 and PNPLA4 also inhibited G(0)/G(1) G(1) to S cell cycle progression, which could thus be regulated by GPL turnover. These results strongly suggest that PNPLA9, -6 and -4 play a key role in GPL turnover and homeostasis in human cells. A hypothetical model suggesting how these enzymes could recognize the relative concentration of the different GPLs is proposed. (C) 2016 Elsevier B.V. All rights reserved.Peer reviewe

Introduction of phospholipids to cultured cells with cyclodextrin

Peer reviewe

Comment on "First-principles study of the influence of (110)-oriented strain on the ferroelectric properties of rutile TiO2"

In a recent article, Gr\"{u}nebohm et al. [Phys. Rev. B 84 132105 (2011), arXiv:1106.2820] report that they fail to reproduce the A2u ferroelectric instability of TiO2 in the rutile structure calculated with density functional theory within the PBE-GGA approximation by Montanari et al. [Chem. Phys. Lett 364, 528 (2002)]. We demonstrate that this disagreement arises from an erroneous treatment of Ti 3s and 3p semi-core electrons as core in their calculations. Fortuitously the effect of the frozen semi-core pseudopotential cancels the phonon instability of the PBE exchange-correlation, and the combination yields phonon frequencies similar to the LDA harmonic values. Gr\"{u}nebohm et al. also attempted and failed to reproduce the soft acoustic phonon mode instability under (110) strain reported by Mitev et al. [Phys. Rev. B 81 134303 (2010)]. For this mode the combination of PBE-GGA and frozen semi-core yields a small imaginary frequency of 9.8i. The failure of Gr\"{u}nebohm et al. to find this mode probably arose from numerical limitations of the geometry optimization approach in the presence of a shallow double well potential; the optimization method is not suitable for locating such instabilities.Comment: 5 page

Dynamics of the Ethanolamine Glycerophospholipid Remodeling Network

Peer reviewe

Aspirin and extended-release dipyridamole versus clopidogrel for recurrent stroke

Background Recurrent stroke is a frequent, disabling event after ischemic stroke. This study compared the efficacy and safety of two antiplatelet regimens — aspirin plus extendedrelease dipyridamole (ASA–ERDP) versus clopidogrel. Methods In this double-blind, 2-by-2 factorial trial, we randomly assigned patients to receive 25 mg of aspirin plus 200 mg of extended-release dipyridamole twice daily or to receive 75 mg of clopidogrel daily. The primary outcome was first recurrence of stroke. The secondary outcome was a composite of stroke, myocardial infarction, or death from vascular causes. Sequential statistical testing of noninferiority (margin of 1.075), followed by superiority testing, was planned. Results A total of 20,332 patients were followed for a mean of 2.5 years. Recurrent stroke occurred in 916 patients (9.0%) receiving ASA–ERDP and in 898 patients (8.8%) receiving clopidogrel (hazard ratio, 1.01; 95% confidence interval [CI], 0.92 to 1.11). The secondary outcome occurred in 1333 patients (13.1%) in each group (hazard ratio for ASA–ERDP, 0.99; 95% CI, 0.92 to 1.07). There were more major hemorrhagic events among ASA–ERDP recipients (419 [4.1%]) than among clopidogrel recipients (365 [3.6%]) (hazard ratio, 1.15; 95% CI, 1.00 to 1.32), including intracranial hemorrhage (hazard ratio, 1.42; 95% CI, 1.11 to 1.83). The net risk of recurrent stroke or major hemorrhagic event was similar in the two groups (1194 ASA–ERDP recipients [11.7%], vs. 1156 clopidogrel recipients [11.4%]; hazard ratio, 1.03; 95% CI, 0.95 to 1.11). Conclusions The trial did not meet the predefined criteria for noninferiority but showed similar rates of recurrent stroke with ASA–ERDP and with clopidogrel. There is no evidence that either of the two treatments was superior to the other in the prevention of recurrent stroke. (ClinicalTrials.gov number, NCT00153062.

Acyl Chain-Dependent Effect of Lysophosphatidylcholine on Endothelium-Dependent Vasorelaxation

Peer reviewe

Rheological and textural properties of sodium reduced salt soluble myofibrillar protein gels containing sodium tri-polyphosphate

The effect of partial replacement of NaCl (50%) with KCl in the presence of sodium tri-polyphosphate (STPP) on the cooking loss (CL), water-holding capacity (WHC), rheological and textural properties of salt soluble myofibrillar proteins (SSMP) gels was investigated. KCl substitution, either alone or in combination with STPP, was found worse than NaCl alone in terms of elasticity of the gels (G′). Both KCl and STPP reduced the CL of the gels. While the gels with replacement of NaCl with KCl had lower (P  0.05) WHC to only NaCl added gels. Substitution of NaCl with KCl resulted in a decrease in hardness of gels whereas STPP addition improved the hardness. Results obtained from the present study suggest that substitution 50% of NaCl with KCl in presence of STPP would be a sound salt reduction alternative in meat systems. Practical Applications: Salt is one of the most crucial ingredients affecting meat products functionality. However, recent research has focused on reducing salt in meat products due to its association with some health problems such as hypertension. Reducing sodium chloride (NaCl) decreases extractability of salt soluble myofibrillar proteins (SSMP), thus, negatively affects functional properties of the meat systems. One of the approaches in reducing salt is to replace NaCl with other chloride salts, the most common of which is potassium chloride (KCl). This study focused on determining the effect of partially substituting of NaCl with KCl in the presence of sodium tri-polyphosphate (STPP) on some functional characteristics of meat SSMP gels. Results indicated that 50% NaCl reduction using KCl and STPP could be achieved with an improvement of textural and functional properties in meat gels. Future research should focus on combined effects of salt substitutes on the rheological and textural characteristics of meat gel systems

Data Management Plans: the Importance of Data Management in the BIG‐MAP Project[]**

Open access to research data is increasingly important for accelerating research. Grant authorities therefore request detailed plans for how data is managed in the projects they finance. We have recently developed such a plan for the EU−H2020 BIG-MAP project—a cross-disciplinary project targeting disruptive battery-material discoveries. Essential for reaching the goal is extensive sharing of research data across scales, disciplines and stakeholders, not limited to BIG-MAP and the European BATTERY 2030+ initiative but within the entire battery community. The key challenges faced in developing the data management plan for such a large and complex project were to generate an overview of the enormous amount of data that will be produced, to build an understanding of the data flow within the project and to agree on a roadmap for making all data FAIR (findable, accessible, interoperable, reusable). This paper describes the process we followed and how we structured the plan