Browsing by Subject "Lipids"

Now showing 1 - 5 of 5

Open Access
Differential effects of nitrogen and sulfur deprivation on growth and biodiesel feedstock production of Chlamydomonas reinhardtii
(2012) Cakmak, T.; Angun P.; Demiray, Y.E.; Ozkan, A.D.; Elibol, Z.; Tekinay, T.
Biodiesel production from microalgae is a promising approach for energy production; however, high cost of its process limits the use of microalgal biodiesel. Increasing the levels of triacylglycerol (TAG) levels, which is used as a biodiesel feedstock, in microalgae has been achieved mainly by nitrogen starvation. In this study, we compared effects of sulfur (S) and nitrogen (N) starvation on TAG accumulation and related parameters in wild-type Chlamydomonas reinhardtii CC-124 mt(-) and CC-125 mt(+) strains. Cell division was interrupted, protein and chlorophyll levels rapidly declined while cell volume, total neutral lipid, carotenoid, and carbohydrate content increased in response to nutrient starvation. Cytosolic lipid droplets in microalgae under nutrient starvation were monitored by three-dimensional confocal laser imaging of live cells. Infrared spectroscopy results showed that relative TAG, oligosaccharide and polysaccharide levels increased rapidly in response to nutrient starvation, especially S starvation. Both strains exhibited similar levels of regulation responses under mineral deficiency, however, the degree of their responses were significantly different, which emphasizes the importance of mating type on the physiological response of algae. Neutral lipid, TAG, and carbohydrate levels reached their peak values following 4 days of N or S starvation. Therefore, 4 days of N or S starvation provides an excellent way of increasing TAG content. Although increase in these parameters was followed by a subsequent decline in N-starved strains after 4 days, this decline was not observed in S-starved ones, which shows that S starvation is a better way of increasing TAG production of C. reinhardtii than N starvation. © 2012 Wiley Periodicals, Inc.
Open Access
Generation of phospholipid vesicle-nanotube networks and transport of molecules therein
(2011) Jesorka, A.; Stepanyants, N.; Zhang H.; Ortmen, B.; Hakonen, B.; Orwar O.
We describe micromanipulation and microinjection procedures for the fabrication of soft-matter networks consisting of lipid bilayer nanotubes and surface-immobilized vesicles. These biomimetic membrane systems feature unique structural flexibility and expandability and, unlike solid-state microfluidic and nanofluidic devices prepared by top-down fabrication, they allow network designs with dynamic control over individual containers and interconnecting conduits. The fabrication is founded on self-assembly of phospholipid molecules, followed by micromanipulation operations, such as membrane electroporation and microinjection, to effect shape transformations of the membrane and create a series of interconnected compartments. Size and geometry of the network can be chosen according to its desired function. Membrane composition is controlled mainly during the self-assembly step, whereas the interior contents of individual containers is defined through a sequence of microneedle injections. Networks cannot be fabricated with other currently available methods of giant unilamellar vesicle preparation (large unilamellar vesicle fusion or electroformation). Described in detail are also three transport modes, which are suitable for moving water-soluble or membrane-bound small molecules, polymers, DNA, proteins and nanoparticles within the networks. The fabrication protocol requires ∼90 min, provided all necessary preparations are made in advance. The transport studies require an additional 60-120 min, depending on the transport regime. © 2011 Nature America, Inc. All rights reserved.
Open Access
Lipid melting transitions involve structural redistribution of interfacial water
(American Chemical Society, 2021-11-03) Schönfeldová, T.; Piller, P.; Kovacik, F.; Pabst, G.; Okur, Halil İbrahim; Roke, S.
Morphological and gel-to-liquid phase transitions of lipid membranes are generally considered to primarily depend on the structural motifs in the hydrophobic core of the bilayer. Structural changes in the aqueous headgroup phase are typically not considered, primarily because they are difficult to quantify. Here, we investigate structural changes of the hydration shells around large unilamellar vesicles (LUVs) in aqueous solution, using differential scanning calorimetry (DSC), and temperature-dependent ζ-potential and high-throughput angle-resolved second harmonic scattering measurements (AR-SHS). Varying the lipid composition from 1,2-dimyristoyl-sn-glycero-3-phosphocholine(DMPC) to 1,2-dimyristoyl-sn-glycero-3-phosphate (DMPA), to 1,2-dimyristoyl-sn-glycero-3-phospho-l-serine (DMPS), we observe surprisingly distinct behavior for the different systems that depend on the chemical composition of the hydrated headgroups. These differences involve changes in hydration following temperature-induced counterion redistribution, or changes in hydration following headgroup reorientation and Stern layer compression.
Open Access
The role of Protein Kinase R in lipotoxicity
(2013) Yağabasan, Büşra
Endoplasmic reticulum (ER) is a central organelle for cellular homeostasis through its myriad of functions including protein and lipid biosynthesis, protein folding and secretion and calcium homeostasis. When protein folding or secretion is disrupted, ER elicits a unique signaling response initiated at its membranes called the unfolded protein response (UPR). UPR attempts to restore cellular homeostasis and survival via reducing unfolded protein levels, however, if this cannot be achieved or the stress is prolonged the UPR could lead to apoptosis. Three specific ER membrane proteins, inositol-requiring enzyme-1 (IRE1), Protein Kinase R-resemble like ER kinase (PERK) and activating transcription factor 6 (ATF6), act as ER stress sensors and initiate distinct but interlaced signaling pathways to restore ER homeostasis. Recently, studies demonstrated that over nutrition, especially high amount of saturated fatty acids or cholesterol in the circulation, leads to the induction of ER stress in metabolic tissues, resulting in the activation of UPR signaling pathways. Furthermore, ER stress was shown to play a causal role in the pathogenesis of metabolic diseases such as obesity, insulin resistance, type 2 diabetes and atherosclerosis. Interferon inducible double strand RNA activated protein kinase R (PKR) is also known to be activated during ER stress. Recent studies showed it can be activated by lipids during ER stress in cells and in metabolic tissues of obese mice. Genetic ablation or inhbition of PKR enhances systemic glucose homeostasis and insulin sensitivity in obesity in rodent models. However, it is not known how PKR becomes activated by overnutrition or by ER stress. In fact, many of the specific cellular components and molecular mechanisms in lipid induced cellular stress or death, namely lipotoxicity, is not completely understood. PKR is one of the serine/threonine kinase that is known to be activated during lipid induced ER stress, but only a few specific downstream substrates are known and these fall short of explaining PKR’s role in lipotoxicity in chronic metabolic disease pathogenesis. PKR also plays a crucial role in activation of inflammasomes through interacting with the inflammasome components. There is a gap in our knowledge regarding PKR’s specific molecular actions in nutrient-induced inflammation and metabolism in chronic metabolic diseases. In this thesis study, my major goal was to develop specific tools to modulate PKR’s activity and search for its specific substrates in lipotoxicity and its role in mediating lipid-induced ER stress response. For this purpose, I developed a novel chemical-genetic approach to specifically modify PKR’s kinase activity during ER stress. In this approach, the bulky sidechain of a gatekeeper amino acid (such as methionine) in the ATP binding cavity of PKR has been altered to a smaller side-chain amino acid (such as glycine) in order to slightly enlarge the cavity to accommodate bulky ATP analogs (activating or inhibiting). This mutant of the PKR has been named the analog sensitive kinase allele (ASKA) of PKR and was shown to utilize normal ATP as well as the bulky ATP analogs in kinase reactions. Furthermore, I demonstrated the specific inhibition of PKR kinase with the inhibitory, bulky ATP analogs such as4-Amino-1-tert-butyl-3-(1’-naphthyl)pyrazolo[3,4-d]pyrimidine (NAPP1) or 4-Amino- 1-tert-butyl-3-(1’-naphthylmethyl)pyrazolo[3,4-d]pyrimidine (1-NMPP1). In order to move one step closer to identification of potential PKR substrates, I also optimized kinase reactions for immunoprecipitated PKR ASKA mutant and visualized several potential downstream substrates in my initial experiments Finally, I studied a unique relationship between two ER stress related kinases IRE1 and PKR in lipid induced ER stress conditions. I observed specific inhibition of IRE1’s endoribonuclease activity with an inhibitor, but not its kinase activity, completely blocks PKR activation by lipids. These findings strongly support hat IRE1’s RNAse activity is necessary for PKR kinase activation by lipids. This function of IRE1 RNAse domain is novel and unsuspected. The future goals of this research should be directed to discovering the RNA mediators of IRE1-PKR coupling and understanding their role in mediating the inflammatory and metabolic pathologies associated with chronic metabolic diseases. In conclusion, in my thesis study, I developed novel chemical-genetic approach to specifically modify PKR kinase activity that could be useful in discovering novel PKR substrates. Based on the preliminary findings in this thesis, PKR appears to have many unidentified substrates regulated during lipid induced ER stress. Furthermore, using the chemical-genetic PKR as a tool as well as several other approaches I demonstrated the existence of a unique, functional relationship between IRE1 and PKR in lipotoxicity. In addition, the results in my thesis shows that IRE1’s endoribonuclease activity is required and sufficient for PKR kinase activation by lipids. These findings and tools developed during my studies can be further utilized for analyzing the specific role of PKR in lipotoxicity, which is important for the health consequences of metabolic diseases.
Open Access
Unfolded protein response regulated mirnas in lipotoxic endoplasmic reticulum stress in macrophages
(2014-07) Terzi, Erdem Murat
The proper functioning and the development of the cell is essential to the fitness of the multicellular organisms - any significant disturbances in cellular mechanisms can lead to a multitude of diseases or death. Among these conditions, the global rise in metabolic diseases like obesity, diabetes and atherosclerosis draw significant research interest focus. Since the prevalence of metabolic disorders in the developed and underdeveloped world is expected to increase further in next decade; understanding the contributing cellular mechanisms is vital for the development of new and effective diagnostic and therapeutic tools against this devastating disease cluster. Among the homeostatic cellular pathways important for health the Unfolded Protein Response (UPR) is highly conserved from yeast to mammals. Aside from most conserved UPR branch Inositol-requiring protein 1(IRE1), the mammalian UPR is composed of three different pathways regulated by IRE1, eukaryotic translation initiation factor 2-alpha kinase 3 (PERK), and activating transcription factor 6 (ATF6). The UPR signaling is activated in response to the accumulation of unfolded or misfolded proteins in ER that leads to endoplasmic reticulum (ER) stress. The goal of the UPR is to re-establish ER homeostasis via inhibition of further protein translation and promoting protein folding. In the case of severe or unresolved ER stress, UPR instead triggers a programmed cell death. Recent studies indicate that noncoding regulatory RNAs such as microRNAs (miRNAs) play important role in both upstream and downstream of the UPR. In this thesis, the regulation of miRNA expression by the different UPR arms are examined in macrophages under lipidinduced or lipotoxic ER stress conditions. The results of PCR array studies of RNA obtained from mouse macrophages stressed with a saturated fatty acid, palmitate (PA) , revealed multiple differentially regulated miRNAs. Among these miRNAs, significantly regulated ones were further examined for their regulation by the different arms of the UPR. Towards this end several complementary approaches were taken: First, significantly regulated microRNAs from microRNA PCR array results were analyzed. Next, macrophages were treated with palmitate after transfection with IRE1 and PERK silencer RNA (siRNA) to assess the role of UPR arms in lipid regulated miRNA regulation and the expression of relevant miRNAs was examined in treated macrophages. As an alternative method, macrophages were treated simultaneously with palmitate and specific inhibitors for IRE1’s endoribonuclease or PERK’s kinase activity. Then miRNA expressions were further examined in IRE1 knock-out mouse embryonic fibroblast (MEF) cell lines transfected with the wild type (WT) IRE or the endoribonuclease domain inactive (RD) mutant of IRE1 to verify the specific regulation of the miRNA by the IRE1’s endoribonuclease activity. As a result, upregulation of miR-2137 expression by palmitate was determined as IRE1-endoribonuclease dependent. Next, potential target mRNAs were examined by the overexpression or knock-down of miR-2137 in macrophages. One possible target mRNA was found to be inositol polyphosphate phosphatase-like 1 (Innpl1) . Aside from miR-2137, miR-33 also showed significant alteration upon PA treatment in macrophages. Since the role of miR-33 in atherosclerosis, obesity and insulin resistance is well established, its expression was studied further in RAW 264.7 macrophage cell line and bone marrow-derived primary macrophages after IRE1 and PERK knock-down with siRNA. ATP-binding cassette, sub-family A (ABC1), member 1 (ABCA1), a known target of miR-33, was investigated as down-stream target of miR-33 in PA treated macrophages, in an IRE1 dependent manner. The results of this study uncovered new UPR regulated miRNAs under lipid stress in macrophages. Excess lipid is one of the prominent causes in metabolic diseases – obesity, atherosclerosis, insulin resistance – and these UPR regulated miRNAs may explain the underlying mechanism behind this set of diseases. Furthermore, the possible gene targets for these miRNAs could be responsible for progression of such conditions. Further studies are needed to reveal the exact mechanisms that can lead to the development of novel therapeutic approaches.