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| 2. |
- Birney, Ewan, et al.
(author)
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Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project
- 2007
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In: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 447:7146, s. 799-816
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Journal article (peer-reviewed)abstract
- We report the generation and analysis of functional data from multiple, diverse experiments performed on a targeted 1% of the human genome as part of the pilot phase of the ENCODE Project. These data have been further integrated and augmented by a number of evolutionary and computational analyses. Together, our results advance the collective knowledge about human genome function in several major areas. First, our studies provide convincing evidence that the genome is pervasively transcribed, such that the majority of its bases can be found in primary transcripts, including non-protein-coding transcripts, and those that extensively overlap one another. Second, systematic examination of transcriptional regulation has yielded new understanding about transcription start sites, including their relationship to specific regulatory sequences and features of chromatin accessibility and histone modification. Third, a more sophisticated view of chromatin structure has emerged, including its inter-relationship with DNA replication and transcriptional regulation. Finally, integration of these new sources of information, in particular with respect to mammalian evolution based on inter- and intra-species sequence comparisons, has yielded new mechanistic and evolutionary insights concerning the functional landscape of the human genome. Together, these studies are defining a path for pursuit of a more comprehensive characterization of human genome function.
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| 3. |
- Alonso-Mori, Roberto, et al.
(author)
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Energy-dispersive X-ray emission spectroscopy using an X-ray free-electron laser in a shot-by-shot mode
- 2012
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In: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 109:47, s. 19103-19107
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Journal article (peer-reviewed)abstract
- The ultrabright femtosecond X-ray pulses provided by X-ray free-electron lasers open capabilities for studying the structure and dynamics of a wide variety of systems beyond what is possible with synchrotron sources. Recently, this probe-before-destroy approach has been demonstrated for atomic structure determination by serial X-ray diffraction of microcrystals. There has been the question whether a similar approach can be extended to probe the local electronic structure by X-ray spectroscopy. To address this, we have carried out femtosecond X-ray emission spectroscopy (XES) at the Linac Coherent Light Source using redox-active Mn complexes. XES probes the charge and spin states as well as the ligand environment, critical for understanding the functional role of redox-active metal sites. K beta(1,3) XES spectra of Mn-II and Mn-2(III,IV) complexes at room temperature were collected using a wavelength dispersive spectrometer and femtosecond X-ray pulses with an individual dose of up to > 100 MGy. The spectra were found in agreement with undamaged spectra collected at low dose using synchrotron radiation. Our results demonstrate that the intact electronic structure of redox active transition metal compounds in different oxidation states can be characterized with this shot-by-shot method. This opens the door for studying the chemical dynamics of metal catalytic sites by following reactions under functional conditions. The technique can be combined with X-ray diffraction to simultaneously obtain the geometric structure of the overall protein and the local chemistry of active metal sites and is expected to prove valuable for understanding the mechanism of important metalloproteins, such as photosystem II.
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| 4. |
- Amrutkar, Manoj, et al.
(author)
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Protein kinase STK25 controls lipid partitioning in hepatocytes and correlates with liver fat content in humans
- 2016
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In: Diabetologia. - : Springer Science and Business Media LLC. - 0012-186X .- 1432-0428. ; 59:2, s. 341-353
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Journal article (peer-reviewed)abstract
- Aims/hypothesis Type 2 diabetes is closely associated with pathological lipid accumulation in the liver, which is suggested to actively contribute to the development of insulin resistance. We recently identified serine/threonine protein kinase 25 (STK25) as a regulator of liver steatosis, whole-body glucose tolerance and insulin sensitivity in a mouse model system. The aim of this study was to assess the role of STK25 in the control of lipid metabolism in human liver. Methods Intracellular fat deposition, lipid metabolism and insulin sensitivity were studied in immortalised human hepatocytes (IHHs) and HepG2 hepatocellular carcinoma cells in which STK25 was overexpressed or knocked down by small interfering RNA. The association between STK25 mRNA expression in human liver biopsies and hepatic fat content was analysed. Results Overexpression of STK25 in IHH and HepG2 cells enhanced lipid deposition by suppressing beta-oxidation and triacylglycerol (TAG) secretion, while increasing lipid synthesis. Conversely, knockdown of STK25 attenuated lipid accumulation by stimulating beta-oxidation and TAG secretion, while inhibiting lipid synthesis. Furthermore, TAG hydrolase activity was repressed in hepatocytes overexpressing STK25 and reciprocally increased in cells with STK25 knockdown. Insulin sensitivity was reduced in STK25-overexpressing cells and enhanced in STK25-deficient hepatocytes. We also found a statistically significant positive correlation between STK25 mRNA expression in human liver biopsies and hepatic fat content. Conclusions/interpretation Our data suggest that STK25 regulates lipid partitioning in human liver cells by controlling TAG synthesis as well as lipolytic activity and thereby NEFA release from lipid droplets for beta-oxidation and TAG secretion. Our findings highlight STK25 as a potential drug target for the prevention and treatment of type 2 diabetes.
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| 5. |
- Amrutkar, Manoj, et al.
(author)
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STK25 is a critical determinant in nonalcoholic steatohepatitis.
- 2016
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In: FASEB journal : official publication of the Federation of American Societies for Experimental Biology. - 1530-6860. ; 30:10, s. 3628-3643
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Journal article (peer-reviewed)abstract
- Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease, and 10-20% of patients with NAFLD progress to nonalcoholic steatohepatitis (NASH) with a high risk of cirrhosis, liver failure, and hepatocellular carcinoma. Despite its high medical importance, the molecular mechanisms controlling progression from simple liver steatosis to NASH remain elusive. We recently identified serine/threonine protein kinase (STK)25 as a critical regulator of ectopic lipid deposition, systemic glucose, and insulin homeostasis. To elucidate the role of STK25 in the development of NASH, we challenged Stk25-knockout and transgenic mice with a methionine and choline-deficient (MCD) diet. We show that Stk25(-/-) mice are protected against MCD-diet-induced NASH, as evidenced by repressed liver steatosis, oxidative damage, inflammation, and fibrosis, whereas Stk25 transgenic mice developed a more severe NASH phenotype, compared with corresponding wild-type littermates. Consistently, NASH features were suppressed in STK25-deficient human hepatocytes cultured in MCD medium, and reciprocally enhanced in STK25-overexpressing cells. We also found a significant positive correlation in human liver biopsies between STK25 expression and NASH development. The study provides evidence for multiple roles of STK25 in NASH pathogenesis and future investigations to address the potential therapeutic relevance of pharmacological STK25 inhibitors in prevention and treatment of NASH are warranted.-Amrutkar, M., Chursa, U., Kern, M., Nuñez-Durán, E., Ståhlman, M., Sütt, S., Borén, J., Johansson, B. R., Marschall, H.-U., Blüher, M., Mahlapuu, M. STK25 is a critical determinant in nonalcoholic steatohepatitis.
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| 6. |
- Bergmann, Uwe, et al.
(author)
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Using X-ray free-electron lasers for spectroscopy of molecular catalysts and metalloenzymes
- 2021
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In: NATURE REVIEWS PHYSICS. - : Springer Nature. - 2522-5820. ; 3:4, s. 264-282
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Research review (peer-reviewed)abstract
- X-ray lasers offer unprecedented capabilities, with their tunable, intense and short X-ray pulses. This Technical Review discusses the current and future use of X-ray lasers for probing molecular catalysts and metalloenzymes and their chemical reactions in real time and under functional conditions. The metal centres in metalloenzymes and molecular catalysts are responsible for the rearrangement of atoms and electrons during complex chemical reactions, and they enable selective pathways of charge and spin transfer, bond breaking/making and the formation of new molecules. Mapping the electronic structural changes at the metal sites during the reactions gives a unique mechanistic insight that has been difficult to obtain to date. The development of X-ray free-electron lasers (XFELs) enables powerful new probes of electronic structure dynamics to advance our understanding of metalloenzymes. The ultrashort, intense and tunable XFEL pulses enable X-ray spectroscopic studies of metalloenzymes, molecular catalysts and chemical reactions, under functional conditions and in real time. In this Technical Review, we describe the current state of the art of X-ray spectroscopy studies at XFELs and highlight some new techniques currently under development. With more XFEL facilities starting operation and more in the planning or construction phase, new capabilities are expected, including high repetition rate, better XFEL pulse control and advanced instrumentation. For the first time, it will be possible to make real-time molecular movies of metalloenzymes and catalysts in solution, while chemical reactions are taking place.
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| 7. |
- Bhowmick, Asmit, et al.
(author)
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Going around the Kok cycle of the water oxidation reaction with femtosecond X-ray crystallography
- 2023
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In: IUCrJ. - : International Union Of Crystallography. - 2052-2525. ; 10:6, s. 642-655
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Research review (peer-reviewed)abstract
- The water oxidation reaction in photosystem II (PS II) produces most of the molecular oxygen in the atmosphere, which sustains life on Earth, and in this process releases four electrons and four protons that drive the downstream process of CO2 fixation in the photosynthetic apparatus. The catalytic center of PS II is an oxygen-bridged Mn4Ca complex (Mn4CaO5) which is progressively oxidized upon the absorption of light by the chlorophyll of the PS II reaction center, and the accumulation of four oxidative equivalents in the catalytic center results in the oxidation of two waters to dioxygen in the last step. The recent emergence of X-ray free-electron lasers (XFELs) with intense femtosecond X-ray pulses has opened up opportunities to visualize this reaction in PS II as it proceeds through the catalytic cycle. In this review, we summarize our recent studies of the catalytic reaction in PS II by following the structural changes along the reaction pathway via room-temperature X-ray crystallography using XFELs. The evolution of the electron density changes at the Mn complex reveals notable structural changes, including the insertion of OX from a new water molecule, which disappears on completion of the reaction, implicating it in the O-O bond formation reaction. We were also able to follow the structural dynamics of the protein coordinating with the catalytic complex and of channels within the protein that are important for substrate and product transport, revealing well orchestrated conformational changes in response to the electronic changes at the Mn4Ca cluster.
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| 8. |
- Bhowmick, Asmit, et al.
(author)
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Structural evidence for intermediates during O2 formation in photosystem II
- 2023
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In: Nature. - : Springer Nature. - 0028-0836 .- 1476-4687. ; 617:7961, s. 629-636
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Journal article (peer-reviewed)abstract
- In natural photosynthesis, the light-driven splitting of water into electrons, protons and molecular oxygen forms the first step of the solar-to-chemical energy conversion process. The reaction takes place in photosystem II, where the Mn4CaO5 cluster first stores four oxidizing equivalents, the S0 to S4 intermediate states in the Kok cycle, sequentially generated by photochemical charge separations in the reaction center and then catalyzes the O–O bond formation chemistry. Here, we report room temperature snapshots by serial femtosecond X-ray crystallography to provide structural insights into the final reaction step of Kok’s photosynthetic water oxidation cycle, the S3→[S4]→S0 transition where O2 is formed and Kok’s water oxidation clock is reset. Our data reveal a complex sequence of events, which occur over micro- to milliseconds, comprising changes at the Mn4CaO5 cluster, its ligands and water pathways as well as controlled proton release through the hydrogen-bonding network of the Cl1 channel. Importantly, the extra O atom Ox, which was introduced as a bridging ligand between Ca and Mn1 during the S2→S3 transition, disappears or relocates in parallel with Yz reduction starting at approximately 700 μs after the third flash. The onset of O2 evolution, as indicated by the shortening of the Mn1–Mn4 distance, occurs at around 1,200 μs, signifying the presence of a reduced intermediate, possibly a bound peroxide.
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| 9. |
- Bogacz, Isabel, et al.
(author)
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Room temperature X-ray absorption spectroscopy of metalloenzymes with drop-on-demand sample delivery at XFELs
- 2023
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In: Pure and Applied Chemistry. - 0033-4545. ; 95:8, s. 891-897
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Journal article (peer-reviewed)abstract
- X-ray crystallography and X-ray spectroscopy using X-ray free electron lasers plays an important role in understanding the interplay of structural changes in the protein and the chemical changes at the metal active site of metalloenzymes through their catalytic cycles. As a part of such an effort, we report here our recent development of methods for X-ray absorption spectroscopy (XAS) at XFELs to study dilute biological samples, available in limited volumes. Our prime target is Photosystem II (PS II), a multi subunit membrane protein complex, that catalyzes the light-driven water oxidation reaction at the Mn4CaO5 cluster. This is an ideal system to investigate how to control multi-electron/proton chemistry, using the flexibility of metal redox states, in coordination with the protein and the water network. We describe the method that we have developed to collect XAS data using PS II samples with a Mn concentration of <1 mM, using a drop-on-demand sample delivery method.
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| 10. |
- Chatterjee, Ruchira, et al.
(author)
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Structural isomers of the S-2 state in photosystem II : do they exist at room temperature and are they important for function?
- 2019
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In: Physiologia Plantarum. - : Wiley-Blackwell. - 0031-9317 .- 1399-3054. ; 166:1, s. 60-72
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Journal article (peer-reviewed)abstract
- In nature, an oxo‐bridged Mn4CaO5 cluster embedded in photosystem II (PSII), a membrane‐bound multi‐subunit pigment protein complex, catalyzes the water oxidation reaction that is driven by light‐induced charge separations in the reaction center of PSII. The Mn4CaO5 cluster accumulates four oxidizing equivalents to enable the four‐electron four‐proton catalysis of two water molecules to one dioxygen molecule and cycles through five intermediate S‐states, S0 – S4 in the Kok cycle. One important question related to the catalytic mechanism of the oxygen‐evolving complex (OEC) that remains is, whether structural isomers are present in some of the intermediate S‐states and if such equilibria are essential for the mechanism of the O‐O bond formation. Here we compare results from electron paramagnetic resonance (EPR) and X‐ray absorption spectroscopy (XAS) obtained at cryogenic temperatures for the S2state of PSII with structural data collected of the S1, S2 and S3 states by serial crystallography at neutral pH (∼6.5) using an X‐ray free electron laser at room temperature. While the cryogenic data show the presence of at least two structural forms of the S2 state, the room temperature crystallography data can be well‐described by just one S2 structure. We discuss the deviating results and outline experimental strategies for clarifying this mechanistically important question.
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