bluuebunny/crossref_metadata_2025 huggingface dataset:
Understanding Plant Energy Sensing and Homeostasis
Authors: Williams Sarah Phoebe , Yen Jiun , Gillaspy Glenda
Published: November 2023

The Sucrose non‐Fermenting Related Kinase 1 (SnRK1) protein family has been shown to play a critical role in metabolic energy regulation and stress signaling. SnRK1, a homolog of yeast Snf‐1 (Sucrose non‐fermenting‐1) and human AMPK (AMP Kinase), is a serine/threonine kinase which is activated by phosphorylation and in turn acts to regulate key metabolic enzymes and transcriptions factors, resulting in the metabolic reprograming of the cell. The SnRK1 gene family contains three members, SnRK1.1, 1.2, and 1.3. The up‐regulation of SnRK1.1 in Arabidopsis has generated plants with enhanced biomass and altered developmental rate. SnRK1.1 over‐expressing (OE) plants grown on soil are smaller than wild‐type plants pre‐flowering, have a delay in flowering, increased biomass post‐flowering, and delayed senescence. The underlying metabolic mechanism of this valuable trait, increased biomass, has not been fully elucidated due to its complexity. In this work, we sought to understand the metabolic changes associated with these phenotypes. In conjunction with Metabolon, Inc., we took a metabolomics approach to measure over 300 metabolites in SnRK1 OE and WT plants. These data were used to address two main questions. The first, Are SnRK1‐induced metabolic changes the same in early development (where growth is suppressed), as compared to later development (where growth is increased)? From these data we found 65% of the metabolites detected in wildtype plants were significantly different as a result of developmental transition suggesting plant metabolism changes with developmental state. While SnRK1 OE alters metabolism across development, it does not do so equally, with the greatest differences observed post‐flowering. SnRK1 OE and wildtype plants were more similar at the pre‐flowering stage, with 54 metabolites elevated and 58 decreased in SnRK1 OE plants as compared to wildtype. In contrast, 116 metabolites were elevated and 140 decreased in post‐flowering SnRK1 OE plants as compared to wildtype. These changes suggest differences in carbohydrate metabolism, lipid composition, nitrogen assimilation and stress response and autophagy pathways. We posed the second question, How does SnRK1 OE alter metabolism, and are changes similar to those seen in carbon starvation or other putative energy‐sensing mutants? The set of metabolic changes were compared to known metabolic and transcriptional profiles of carbon starved plants. There was significant overlap between these data sets and the metabolic changes observed in a SnRK1 OE plant at both pre‐ and post‐flower stages. Overall, these data suggest that SnRK1 overexpression is likely inducing carbon starvation metabolic pathways.
Independent Measurements of Lipids in Mixed Cell Populations
Authors: Stoeckman Angela , Estrada Katerina , Wolins Nathan
Published: November 2023

Metabolic diseases such as type 2 diabetes, atherosclerosis, and non‐alcoholic fatty liver disease (NAFLD) are a major cause for concern worldwide with one in three being diagnosed with NAFLD in the United States. NAFLD is a better indicator of metabolic disease than obesity and it is likely that how fat is deposited at the cellular level, rather than the organ level, will be an even better predictor of metabolic disease than obesity or NAFLD alone. In each of the aforementioned metabolic diseases, there is a pathological increase in the amount of lipid stored in specific cell types such as adipocytes, myocytes, macrophages, or hepatocytes leaving the cell or tissue vulnerable to a multitude of cellular stresses. The stored lipids are predominantly triglycerides and cholesterol esters and are sequestered into organelles called lipid droplets. These organelles have the dynamic ability to change in size based on the needs of the organism. Therefore, the ability to accurately assess changes in the amount of lipid in these droplets is essential. Traditionally, methods to quantify lipids have included cell isolation and lysis followed by organic lipid extraction and measurement of triglyceride mass or cell microscopy techniques where lipids are visualized through the use of fluorescent stains. These current techniques are time‐consuming and cannot differentiate between unique lipid levels within the same sample. Here, we demonstrate the use of a lipophilic fluorochrome in a flow cytometry assay as a valid method for quantifying lipid droplets in multiple cell types as a direct comparison against traditional methods. We observed a dose‐dependent increase in mean fluorescence intensity in multiple cell types treated with the unsaturated fatty acid oleate. There was a positive correlation between our flow cytometry technique and the triglyceride assay and fluorescence microscopy techniques. The advantage to our flow cytometry‐based approach was improved sensitivity obtained in a fraction of the time as compared to traditional methods. Also, we highlight the ability of our method to assess differing levels of lipid droplets within a mixed cell population of AML‐12 mouse hepatocytes and 293 human kidney cells fed different amounts of oleate as well as within human peripheral blood mononuclear cells. Further, when gating only on lymphocytes within a mixture of human peripheral blood mononuclear cells, subpopulations could be resolved by lipid levels. Using flow cytometry for the quantitation of lipid droplets will allow researchers and clinicians to obtain accurate data quickly, reproducibly, and within human samples which may contain a mixed population of cells each with distinct levels of lipid stores.
Characterization of kinetic parameters of the lysophospholipase PldB using a liquid chromatography mass spectrometry based in vitro assay
Authors: Elizalde Gabriel , Garrett Teresa A
Published: November 2023

Headgroup‐acylated glycerophospholipids (HAGPLs), such as acyl phosphatidylglycerol (acyl PG) and N‐acyl phosphatidylethanolamine, are a class of low‐abundance lipids that may play a role in Escherichia coli cell division. Lysophospholipase L2, also known as PldB, is an acyltransferase found in E. coli known to catalyze the formation of acyl PG. We seek to fully understand the substrate specificity of PldB to ultimately determine what contributes to PldB being an acyltransferase versus a phospholipase. Previous research suggests that PldB catalyzes the transfer of the acyl chain from 2‐acyl lysophospholipids to phosphatidylglycerol to form acyl PG. In order to characterize PldB's substrate specificity, a six‐histidine tagged variant of PldB has been constructed and overexpressed using the pET overexpression system. Cells induced to overexpress PldB are lysed, membranes isolated and PldB solubilized from the membranes with high concentrations of potassium chloride. The solubilized PldB is then purified to homogeneity from the resulting mixture with Ni‐affinity chromatography followed by gel‐filtration chromatography. This purified PldB will be used to determine the Km and kcat for PldB's reaction with a variety of acyl donors and acceptors using a liquid chromatography mass spectrometry (LC/MS) based assay currently under development.

Support or Funding Information

National Science Foundation Research at Undergraduate Institutions Grant #1516805 and #1152463, and National Science Foundation Major Instrumentations Grant #1039659
Structure of human Niemann‐Pick C1 (NPC1) protein and NPC1‐NPC2 complex
Authors: Li Xiaochun
Published: November 2023

Cholesterol, essential for all animal life, plays a vital role for maintenance of membrane strength and permeability, also serves as a precursor for the biosynthesis of lipoprotein, steroid hormones, bile acid and vitamin D. Our major cholesterol sources are endogenous biosynthesis and diet uptake. Egress of LDL‐derived cholesterol from lysosomes requires two lysosomal proteins, integral membrane protein NPC1 and soluble NPC2. Mutations on NPC1 and NPC2 can cause the inborn errors Niemann‐Pick disease, type C, which lead to the early death of the patients; remarkably. We reported the structure of NPC1 at 3.3Å resolution, which reveals internal two‐fold pseudo symmetry along TMs 2–13, and two structurally homologous domains that protrude 60Å into the lysosomal lumen. The sterol‐sensing domain (SSD) of NPC1 presents an outward‐open conformation and has a very cavity, which might transport/sense the cholesterol through the limiting membrane. Two luminal domains [Middle lumenal Domain (MLD) and C‐terminal Domain (CTD)] shared the similar fold. We also determined the 2.4Å resolution crystal structure of a complex of human NPC1‐MLD and NPC2 bearing bound cholesterol‐3‐O‐sulfate. NPC1‐MLD utilizes two protruding loops to bind NPC2, analogous to its interaction with the primed Ebola virus glycoprotein. These works present the atomic resolution models for cholesterol biogenesis and trafficking, provide the molecular insights into cholesterol transfer in lysosomal lumen and pathogenesis of Niemann‐Pick disease, type C.

Support or Funding Information

Life Sciences Research Foundation
Substrate Specificity and Unusual Reaction Mechanism of the Sterol 4‐Methyltransferase in Caenorhabdtis elegans
Authors: Fisher Paxtyn Michael , Zhou Wenxu , Miller Matthew B. , Shen Yun , Shi Huazhong , Nes W. David
Published: November 2023

Studies on the biosynthesis of sterols in animals have been the subject of Nobel Prizes. In vertebrates, cholesterol biosynthesis proceeds from lanosterol to sterol intermediates methylated at the 4‐postion (Nes, W. D. (2011) Chem. Rev. 111, 6423–6451). These compounds typically contain a double bond at the 8‐position and originate by the removal of C4‐methyl from lanosterol. In contrast, the invertebrate worm C. elegans converts cholesterol to C4‐methylsterol end products that possess a double ‐bond at the 7‐ or 8(14)‐position. The C4‐methyl addition is catalyzed by sterol C4‐methyltransferase (4‐SMT) (Hannich, J. T. et al. (2009) Dev. Cell 16, 833–843). There is speculation that C4‐methyl sterols are produced through a route involving direct methylation of lathosterol (cholest‐7‐enol) to form lophenol (4‐methyl cholest‐7‐enol). There is support for this proposal as C3‐methyl hopanoids are considered to be formed by a radical mechanism of direct methylation. We report here that C. elegans, a sterol auxotroph that converts dietary cholesterol or cholestanol to 4‐methyl sterols, when fed cholest‐3‐one (I) or cholest‐7‐3‐one (II) convert these nutrients to 4‐methyl cholestanol and 4‐methyl cholest‐7‐enol, respectively, by a hypothetical 2‐step metabolism involving a 4‐SMT and 4‐reductase enzyme. Using a cloned 4‐SMT functionally expressed in E. coli and an assay system developed for the sterol C24‐methyltransferase (24‐SMT) that includes SAM or D3‐SAM, we observed that I converts to 4‐methyl cholest‐3‐one while II converts to C4‐methyl cholest‐7‐one or C4‐D3‐methyl‐cholest‐7‐one, respectively. Product identities were confirmed by GC‐MS analysis and synthesis. The results show the 4‐SMT has a broad substrate requirement and reaction of lathosterone (II) in the presence of SAM generates a 3‐keto‐4‐enol intermediate that is regio‐ and stereo‐specifically methylated at C4 affording lophenone, a 4α (equatorial)‐methyl 3‐keto sterol. Notably, the amino acid sequences of 4‐SMT and 24‐SMT share approximate 30 % identity and the 4‐SMT contains all the substrate binding segments for sterol and SAM identified in 24‐SMT. Alternatively, a bioinformatic search for a radical SAM motif identified in bacteria genomes fails to uncover an equivalent binding site in 4‐SMT. Thus, we conclude that the reaction catalyzed by C. elegans 4‐SMT is phyla‐specific and proceeds by a tightly coupled keto‐enol‐methylation mechanism which is different from the reaction catalyzed by 24‐SMT that proceeds by a coupled methyl addition‐proton elimination mechanism.
Ceramide‐1‐Phosphate: Characterizing a Fluorescent Lipid and Discovering New Binding Proteins
Authors: Shirey Carolyn M. , Stahelin Robert V.
Published: November 2023

Sphingolipids are a class of biomolecules that play key roles in cellular signaling and membrane trafficking, with main players including sphingosine, sphingosine‐1‐phosphate (S1P), ceramide, and ceramide‐1‐phosphate (C1P). Specifically, C1P has been shown to play a role in important intra‐ and extracellular events such as cell proliferation, phagocytosis, inflammation, tumor metastasis, and migration of macrophages. Despite the identification of several C1P binding proteins such as cytosolic phospholipase A2α (cPLA2α), Annexin A2, diacylglycerol kinase γ, tumor necrosis factor‐α converting enzyme (TACE), and prostaglandin D synthase, there are still many gaps to fill in our understanding of C1P binding proteins and how these cellular interactions are facilitated. Therefore, we characterized a fluorescently labeled C1P for its in vitro and cellular activity, and sought to identify more C1P binding proteins. Here, we investigated a new fluorescently labeled C1P, called TopFluor‐C1P, to see if it could mimic C1P naturally found in mammalian cells. TopFluor‐C1P proved to be non‐toxic to cells, localize to the trans‐Golgi Network and plasma membrane, and successfully recruit the translocation of cPLA2α, as previous work has shown with native C1P. We also used fluorescence recovery after photobleaching (FRAP) to determine the mobile fraction and diffusion coefficient of TopFluor‐C1P at the plasma membrane to understand C1P membrane dynamics. This revealed the diffusion coefficient of C1P is less than other signaling lipids such as phosphatidylserine and cholesterol, and more similar to that of other sphingolipids. From our studies we determined TopFluor‐C1P is a valuable mimetic that can be further utilized to study many important biophysical and cellular questions regarding C1P properties in membranes and its role in lipid‐lipid and lipid‐protein interactions. In addition we aimed to discover the C1P binding proteome. Therefore, we performed an IP assay from A549 lung adenocarcinoma cells using lipid‐coated beads. The mass spectrometry analysis of lipid phosphomonoesters identified 212 C1P, 280 ceramide, 588 S1P, and 449 phosphatidic acid binding proteins. From this data we were were able to identify proteins that were found among all four structurally similar lipids, or were specific to only one lipid. We also used the Gene Ontology consortium and MetaCore pathways analysis software to further identify the molecular function, cellular localization and pathways involved in C1P binding proteins. Identifying these direct effectors of C1P will aid us in discovering C1P lipid‐protein binding characteristics, and help us better understand the role of C1P within the cell and in disease and cancer metabolism.

Support or Funding Information

This material is based upon work supported by the National Science Foundation Graduate Research Fellowship under Grant No. DGE‐1313583.
Disruption of Sphingolipid Biosynthesis Blocks Phagocytosis of Candida albicans
Authors: Tafesse Fikadu G.
Published: November 2023

The ability of phagocytes to clear pathogens is an essential attribute of the innate immune response. The role of signaling lipid molecules such as phosphoinositides is well established, but the role of membrane sphingolipids in phagocytosis is largely unknown. Using a genetic approach and small molecule inhibitors, we show that phagocytosis of Candida albicans requires an intact sphingolipid biosynthetic pathway. Blockade of serine‐palmitoyltransferase (SPT) and ceramide synthase‐enzymes involved in sphingolipid biosynthesis‐by myriocin and fumonisin B1, respectively, impaired phagocytosis by phagocytes. We used CRISPR/Cas9‐mediated genome editing to generate Sptlc2‐deficient DC2.4 dendritic cells, which lack serine palmitoyl transferase activity. Sptlc2−/− DC2.4 cells exhibited a stark defect in phagocytosis, were unable to bind fungal particles and failed to form a normal phagocytic cup to engulf C. albicans. Supplementing the growth media with GM1, the major ganglioside present at the cell surface, restored phagocytic activity of Sptlc2−/− DC2.4 cells. While overall membrane trafficking and endocytic pathways remained functional, Sptlc2−/− DC2.4 cells express reduced levels of the pattern recognition receptors Dectin‐1 and TLR2 at the cell surface. Consistent with the in vitro data, compromised sphingolipid biosynthesis in mice sensitizes the animal to C. albicans infection. Sphingolipid biosynthesis is therefore critical for phagocytosis and in vivo clearance of C. albicans.

Support or Funding Information

This work was funded by The Netherlands Organization for Scientific Research (FGT), and the National Institute of Health grant AI087879 (HLP).
PKC‐δ Mediates Sphk2 Activation And Histone Acetylation In Pseudomonas aeruginosa‐Induced Lung Inflammation
Authors: Ebenezer David Lenin , Fu Panfeng , Ha Alison Wing‐Zi , Natarajan Viswanathan
Published: November 2023

Pseudomonas aeruginosa (PA), a gram‐negative opportunistic pathogen, the leading cause of inflammation‐driven gradual deterioration of the lung function among cystic fibrotic patients, also causes pneumonia in immunosuppressed and mechanically ventilated patients. Our research intends to understand the role of nuclear Sphingolipids in modifying the epigenetic landscape in PA–induced lung inflammation. Previously, we have shown that Sphingosine kinase 2 (SphK2) generated nuclear Sphingosine‐1‐Phospahate (S1P), a simple bioactive sphingolipid, is an epigenetic co‐regulator of PA‐induced lung inflammation, as evident from our murine model and in vitro studies. Recently, we have identified a novel role of Protein Kinase C‐δ (PKC‐δ) in mediating the PA‐induced phosphorylation of Sphingosine Kinase 2 and histone acetylation. Mouse alveolar Type 2 cells challenged with PA induced phosphorylation of PKC‐δ (S 299), which is necessary for its activation and subsequent translocation to nucleus. PA‐induced activation of PKC‐δ also stimulated nuclear SphK2 phosphorylation in mouse lung type II cells. The PKC inhibitor, bisindolylmaleimide, blocked PA‐induced Histone H3 & H4 acetylation in mouse lung epithelial cell line (MLE‐12). Further, PA‐induced SphK2 phosphorylation and histone acetylation was blocked by small interfering RNA (siRNA) silencing of PKC‐δ in MLE‐12 cells. PA‐induced PKC‐δ activation also resulted in elevated mRNA expression of inflammatory cytokines, IL‐6 and TNF‐α, which was decreased significantly by overexpression of adenoviral dominant negative PKC‐δ mutant in MLE‐12 cells, suggesting a key role for PKC‐δ in regulating inflammatory cytokines. Furthermore, use of dominant negative PKC‐δ mutant also suppressed PA‐induced IL‐6 secretion, suggesting the secondary effector role of PKC‐δ in regulating SphK2 mediated down stream expression of inflammatory cytokines. These studies underscore the role of PKC‐δ in regulating PA‐induced inflammation via SphK2 phosphorylation, and increased histone acetylation suggesting that targeting PKC‐δ could ameliorate the bacterial inflammation of the lung.

Support or Funding Information

This work was supported by NIH P01 HL 98050 to VN
Monitoring Live Cell Membrane Lipid Encounter Dynamics with DNA Probe
Authors: You Mingxu
Published: November 2023

Lipids are ubiquitous membrane molecules that broadly involved in regulating signal transduction. Instead of homogeneously distributed, several lipid molecules clustered to form lipid rafts on cell membranes. These lipid rafts separate cell membranes into small pre‐organized compartmentalization. Abnormal level and interaction of lipids has been implicated in the pathogenesis of cancers, lysosomal storage disorders, peripheral neuropathies and secretory diarrhea. The disruption of lipid‐lipid and lipid‐protein interactions is important for pathogenesis of these lipid‐involved diseases. However, studying transient lipid encounter kinetics, especially on live cell membrane, remains a technical challenge. We have developed a DNA‐based probe to monitor lipid encounter kinetics on live leukemia cell membrane. A specific DNA toehold‐mediated strand displacement reaction transduces transient membrane encounter events into readable cumulative fluorescence signals. Our data indicates that previously untraceable cell membrane interaction kinetics among model diacyllipids, cholesterols and tocopherols can now be monitored with standard fluorescence microscopy and flow cytometry. We believe this DNA probe can be potentially used to study lipid encounter rates and preferences during various cell membrane signaling events.
OlyA – A Tool to Study Sphingomyelin‐Cholesterol Interactions in Plasma Membranes
Authors: Endapally Shreya , Radhakrishnan Arun
Published: November 2023

Sphingomyelin (SM) and cholesterol are lipids that are critical for the function of animal cells. Interplay between these two lipids is important for maintaining integrity of the myelin sheath that encases axonal cells and for maintaining functionally distinct pools of cholesterol in the plasma membranes of fibroblast cells. The nature of the interaction between SM and cholesterol has been long debated, with proposals ranging from complexes of the two lipids to no specific interaction between these two lipids.

Here, we have developed a tool to examine the interaction between SM and cholesterol in animal cell membranes. Our tool is derived from a fungal toxin, ostreolysin A (OlyA). We find that OlyA binds to membranes of CHO‐K1 hamster cells when they contain both SM and cholesterol, but not when levels of either lipid are diminished by treatment with sphingomyelinase (which degrades SM) or cyclodextrin (which depletes cholesterol). In model membranes, OlyA shows no binding to 1, 2‐dioleoyl‐sn‐glycero‐3‐phosphocholine (DOPC) – cholesterol membranes even when the cholesterol concentration exceeds 50 mole%. Also, OlyA shows no binding to SM‐containing membranes when epicholesterol (a diasteromer of cholesterol) is present at concentration exceeding 50 mole%. These observations combined with direct binding experiments have given us insights into OlyA's dual lipid specificity and into how SM and cholesterol are organized in membranes.

Support or Funding Information

This work was supported by the Welch Foundation (I‐1793) and the American Heart Association (12SDG12040267).
Investigating Phospholipid Binding Residues in the C Terminus of Ebola Virus Matrix Protein, VP40
Authors: Budicini Melissa , Johnson Kristen , Stahelin Robert V.
Published: November 2023

Introduction

Ebola virus (EBOV) is a lipid‐enveloped virus that causes hemorrhagic fever and a fatality rate of 50–90%. Though EBOV only has 7 genes in its genome, one, VP40 is the main driver of viral egress. VP40 localizes to the plasma membrane where it forms virus like particles (VLPs). While the mechanism of the viral egress is not completely understood, previous studies have found that phospholipids phosphatidylserine (PS) and phosphatidylinositol 4,5‐bisphosphate (PIP2) are required for plasma membrane localization, self‐oligomerization, VLP formation and VLP budding from the cell. VP40 transforms through self‐oligomerization from a dimer to a hexamer and eventually a longer filament. PS likely induces the dimer to hexamer transition while PIP2 binding is required for stability of larger oligomers (n=12 or more). In an effort to understand the process of VP40 oligomerization, we have identified a likely PIP2 binding pocket on the C terminus of VP40; the C terminus of VP40 is hypothesized to interact with the PM. This pocket shares structural similarity to the PIP2 binding pocket of viral matrix protein HIV‐GAG from HIV‐1.

Objective

We are mutating potential binding residues and control residues to alanine on the C terminus of VP40 in order to understand the interactions between VP40, PIP2, and localization to the plasma membrane.

Methods

Site Directed Mutagenesis, Confocal Laser Scanning Microscopy (CLSM), Liposome Pelleting Assays (LPA), and Transmission Electron Microscopy (TEM).

Results

VP40 PM localization can be blocked by elimination of phospholipid substrates through various assays in live COS‐7 cells. We are now investigating how VP40 binds to phospholipids. We have made several mutations with site directed mutagenesis and expressed or purified each in live cells for CLSM, or for lipid binding assays (LPA, TEM). We have identified several amino acids in the proposed binding pocket that are required for PI(4,5)P2 binding and oligomerization.

Conclusions

Phospholipid‐focused assays in live cells and in vitro assays reveal residues in the C‐terminal domain of VP40 that are required to bind to PI(4,5)P2. We aim to better understand this interaction and it's role in oligomerization through future assays.

Support or Funding Information

NIH AI081077
Binding Interaction of Lipid‐bound ApoA–I with Lipopolysaccharides and Phosphatidylglycerol
Authors: Haeri Bahareh , Weers Paul M.M.
Published: November 2023

Apolipoprotein A‐I (apoA‐I) is a major component of high‐density lipoprotein (HDL), an anti‐antherogenic complex responsible for reverse cholesterol transport from peripheral tissues to liver, and mediates several essential metabolic functions related to heart disease. Human apoA‐I is 28 kDa protein found in lipid‐free and lipid‐bound forms. Previous studies have shown that lipid‐free apoA‐I binds and neutralizes lipopolysaccharides (LPS), which are major constituents of the outer bacterial membrane of gram‐negative bacteria, therefore reducing endotoxic effects. In addition, the protein destabilizes bilayers of negatively charged phosphatidylglycerol (PG), a phospholipid found in the inner bacterial membrane, which may result in cell lysis. However, the majority of apoA‐I is found in the lipid‐bound form as HDL; therefore binding interactions with LPS and PG were studied. To this end reconstituted HDL (rHDL) was produced with 1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphocholine (POPC) and apoA‐I, which was isolated by KBr density ultracentrifugation. rHDL was analyzed by native PAGE and showed three distinct lipid‐protein complexes of 246, 203, and 149 kDa. To compare the binding of lipid‐free and lipid bound apoA‐I to LPS, non‐denaturing PAGE was employed. This showed that LPS binding was decreased for rHDL in comparison to lipid‐free apoA‐I. Both forms of apoA‐I were able to disaggregate LPS micelles, as shown by LPS carbohydrate staining of native PAGE. To investigate binding to PG, unilamellar PG vesicles with encapsulated calcein were prepared by extrusion. Calcein is a fluorescent dye and used as an indicator of lipid vesicle leakage, and is self‐quenched at a concentration above 70 mM. Binding to the vesicles was measured by the increase in calcein fluorescence, which was released upon protein‐vesicle binding. Lipid‐free apoA‐I induced the release of 60 to 70% calcein from PG vesicles; however, calcein release was reduced to 20 to 30% for rHDL. These results show that when apoA‐I is in the lipid‐bound state, binding interaction with LPS and PG bilayer vesicles was decreased significantly. Thus in order to be most effective as an antimicrobial protein, apoA‐I needs to be in the lipid‐free conformation.

Support or Funding Information

This research was supported by a grant from the National Institutes of Health (NIGMS #GM089564).
Computational Modeling of Human Fatty Acid Transport Protein 2
Authors: Perez Vincent Michael , Puniya Bhanwar Lal , Helikar Tomáš , DiRusso Concetta C , Black Paul N
Published: November 2023

Fatty acid transport protein 2 (FATP2) functions in the uptake and trafficking of fatty acids through vectorial acylation and belongs to the family of the solute carrier 27A proteins (SLC27A), which have been classified based on their ability to transport exogenous fatty acids into the cell. The FATP2 knockout mouse has depressed serum triglyceride levels and a reduced ability to assimilate dietary fatty acids suggesting a key role in fatty acid transport across the intestinal epithelium. To more fully understand the role of FATP2, we have developed in silico studies to inform the activity of this protein in vivo. Two techniques were employed in these studies: [1] Genome‐scale metabolic modeling, and [2] Ligand docking using a predicted three‐dimensional structure of FATP2. The human genome scale metabolic network model, Recon 2.2, was used to test systematic impacts of FATP2 perturbations on metabolism. Preliminary predictions using Recon 2.2 revealed that knocking out FATP2 is predicted to alter the metabolic flux of 436 reactions (215 up and 221 down). These reactions are encoded by 265 genes. Of interest, 22 of these genes are involved in fatty acid metabolism, 5 of which had increased function and 17 had decreased function after FATP2 perturbation. In addition, we completed preliminary ligand docking studies using the predicted structure of FATP2 retrieved from the Phyre2 server. Using this approach, the substrate binding sites within FATP2 (ATP, long chain fatty acid, and CoA) were predicted to be distinct, yet adjacent to core of the enzyme that may facilitate both substrate binding and product release. Collectively, predicted metabolic flux and ligand binding sites results within FATP2 provide key information that can be tested in vivo to better understand protein‐mediated transport of exogenous fatty acids.

Support or Funding Information

This work is funded by the Agricultural Research Division and Office of Research and Economic Development at the University of Nebraska.
The Ebola Virus Matrix Protein and Membrane Fluidity
Authors: Johnson Kristen A , Stahelin Robert V
Published: November 2023

Introduction

While the Ebola virus is considered a rare and neglected disease, it received worldwide attention during the 2014–15 epidemic. This recent outbreak was eleven times larger than all previous Ebola virus outbreaks combined, taking a large toll on the lives and economies of Western Africa. Unfortunately, there are still no FDA approved therapeutics or vaccines for Ebola virus treatment. The most abundant protein of the virus, which was also the most conserved in amino acid sequence during the course of the outbreak, is the matrix protein VP40. VP40 is sufficient to form virus like particles from human cells independent of other viral proteins and has been suggested to be viable therapeutic target. We aim to better understand how VP40 is able to interact with lipid membranes consisting of different glycerophospholipids and sphingolipids and bend them to form the virus particle. The Stahelin lab has found that VP40 requires the anionic lipids phosphatidylserine (PS) and phosphatidylinositol (4,5)bisphosphate (PI(4,5)P2) at the plasma membrane for proper assembly. We are now investigating if the physical property of membrane fluidity plays a roll in VP40 function.

Objective

We are implementing several assays to investigate the role of membrane fluidity in VP40 membrane binding, oligomerization, and membrane bending.

Methods

Confocal Laser Scanning Microscopy (CLSM), Number and Brightness analysis (N&B), Liposome Pelleting Assays, and Transmission Electron Microscopy (TEM).

Results

VP40 assembly is sensitive to membrane fluidity and has greater binding to ordered membranes than disordered membranes. When membrane fluidity is changed in cells, VP40 localization and large oligomers at the plasma membrane disassemble. We have identified FDA approved drugs that modulate membrane fluidity and are testing their efficacy against VP40 function.

Conclusions

This project aims to better understand VP40 membrane binding and bending functions in relation to membrane fluidity. We have observed that VP40 function is sensitive to changes in membrane fluidity in vitro and in live cells. We hope that a deeper understanding of VP40 dependency on membrane fluidity will allow for expedited drug discovery or reapplication of FDA Approved drugs.

Support or Funding Information

Financial Support: NIH AI081077
The Yeast Homolog of the Mammalian Oncogene, Bax Inhibitor‐1, Regulates the Unfolded Protein Response by Altering the ER Microenvironment.
Authors: Benko Savannah , Eagan David , Alisch Joseph , Brown Melissa , McCarthy Morgan , Cavedon William , O'Brien James , Ritzer Lukas , Berry B. Michael , Austriaco Nicanor
Published: November 2023

Bax inhibitor‐1 (BI‐1) is an anti‐apoptotic gene whose expression is upregulated in a wide range of human cancers. The yeast homolog of BI‐1, yeast Bax inhibitor‐1 (BXI1), encodes a protein that belongs to the Bax Inhibitor (TMBIM) family of proteins. The crystal structure of a prokaryotic member of the family, BsYetJ, has revealed that the Bax inhibitor proteins are pH sensitive calcium leaks. Our studies have shown that yeast Bxi1p is localized to the ER and is involved in the unfolded protein response (UPR) that is triggered by ER stress. We have recently found that Bxi1p regulates the UPR downstream of endonuclease Ire1p clustering but upstream of HAC1 mRNA splicing. Studies with an ER localized calcium reporter and an ER redox reporter suggest that Bxi1p accomplishes this by altering the ER microenvironment.

Support or Funding Information

Our laboratory is supported by grant NIGMS R15 GM110578, awarded to N. Austriaco.
ER stress and molecular targets of platinum anticancer compounds
Authors: Cunningham Rachael , DeRose Victoria
Published: November 2023

Platinum anticancer compounds have been reported to induce ER stress responses in yeast and mammalian cancer cell lines. In this work, using modified ‘click’‐enabled Pt reagents, localization of Pt compounds in treated cells has been followed using post‐treatment fluorescent labeling. Platinum localization to both nucleoli and the ER are observed in Pt‐treated cancer cell lines. The ER stress response has been followed using Xbox1 and/or HAC1 splicing in response to platinum treatment. Finally, platinum association with proteins has been assessed using post‐treatment conjugation to biotin followed by pulldown and MS analysis. Taken together, the results of these studies show that treatment with platinum compounds induces an ER stress response, and support the hypothesis that direct targeting of ER proteins plays a role in this response.

Support or Funding Information

Support form the American Heart Association (predoctoral to R.C.) and the National Science Foundation (CHE‐CLP to VJD) is gratefully acknowledged.
Regulation of the mitochondrial protein quality control factor VMS1
Authors: Fredrickson Eric K. , Nielson Jason R. , Hill Christopher P. , Rutter Jared
Published: November 2023

Mitochondrial function is constantly threatened by damaging agents, including mitochondria‐derived reactive oxygen species. This stochastic burden damages mitochondria, which left unchecked leads to mitochondrial dysfunction. The detrimental consequences of mitochondrial dysfunction are highlighted by its presence in many human diseases including cancer, Parkinson's Disease, and diabetes. To prevent mitochondrial dysfunction, cells have evolved mitochondrial quality control mechanisms, which include protein degradation. A critical component in mitochondrial protein quality control degradation is the protein VCP/Cdc48‐associated Mitochondrial Stress‐responsive (Vms1), which together with Cdc48, translocates to mitochondria under stress. Through the action of Cdc48, an ATPase critical for ER‐associated degradation, Vms1 promotes the proteasomal degradation of proteins embedded in the mitochondrial outer membrane. Deletion of VMS1 results in oxidative stress that leads to a shortened lifespan in yeast and nematodes, which is highly suggestive of mitochondrial dysfunction. Mitochondrial localization of the Cdc48/Vms1 complex depends on a highly conserved Mitochondrial Targeting Domain (MTD), which is inhibited by an intramolecular interaction with the N‐terminal Leucine Rich Sequence (LRS) of Vms1 in unstressed conditions. We aimed to further our understanding of the stress‐responsive localization of Vms1 to mitochondria through structural and biochemical studies. Toward this goal we have: (1) identified a modified mitochondrial sphingolipid that is necessary and sufficient for Vms1 mitochondrial localization, and (2) determined the structure of Vms11–417 (2.7Å, 25% R‐free), which includes the MTD/LRS interaction. These data suggest a model in which the novel MTD fold binds the sphingolipid receptor head group in a pocket that is masked in the auto inhibited state by the LRS and becomes accessible following a conformational change. This model is being tested by structure‐based mutagenic, biochemical, and genetic experiments.

Support or Funding Information

NIH GM115129

AHA Postdoctoral Fellowship 14POST20380216
Effects of Deceased Mitochondrial Ca2+ Uptake in an in vitro Model of Parkinson's Disease
Authors: de la Rosa‐González Nicole , Lee Amy , Joiner Mei‐ling
Published: November 2023

Parkinson's disease is characterized by a progressive and selective loss of dopaminergic neurons in the substantia nigra pars compacta (SNc) of the midbrain. All cells use Ca2+ among other ions to produce signals that regulate cell function. Ca2+ signals in neurons alter electrical activity and stimulate release of neurotransmitters. One of the roles of mitochondria is to help maintain appropriate concentrations of Ca2+ within the cell. The mitochondrial calcium uniporter (MCU) allows Ca2+ into the mitochondria matrix. Under stress conditions, more cytosolic Ca2+ is present in the cell, which can cause excessive Ca2+ uptake by the MCU. It is thought that this excess mitochondrial Ca2+ uptake causes mitochondrial dysfunction which may promote Parkinson's disease. A mechanism that regulates excess mitochondrial Ca2+ uptake in dopaminergic cells might reveal a new target for therapeutic strategies to halt the progressive degeneration of SNc neurons in Parkinson's disease. One such mechanism would be to inhibit MCU, then test if this inhibition protects the cell from high levels of cytosolic Ca2+. For this experiment, we inhibited the activity of the MCU by expressing a dominant‐negative (DN) MCU variant, restricting Ca2+ uptake into the mitochondria matrix of SH‐SY5Y cells, a dopaminergic cell line, that are treated with the dopaminergic neurotoxin, 6‐hydroxydopamine (6‐OHDA). Resulst showed that the expression of DN‐MCU does not reduce cell death, and in this case, is promoting cell death. The expression of DN‐MCU may generate persistent excessive levels of cytosolic Ca2+, generating stress conditions that ultimately lead to cell death and the promotion of Parkinson's disease.

Support or Funding Information

Thanks to Amy Lee's lab for the intensive training during the summer of 2016. Funding for this project was provided by the Summer Research Opportunities Program, a Carver Trust Collaborative Pilot Grant and, the Ponce Research Initiative for Scientific Enhancement (RISE) under the grant # 2R25GM096955.
Cardiac Mitochondrial Phospholipid Acyl Chains are Remodeled in Murine Obesity but do not Impair Supercomplex Formation, Respiratory Enzyme Activity, or Respiration
Authors: Sullivan Elizabeth M. , Fix Amy , Crouch Miranda J. , Sparagna Genevieve C. , Zeczycki Tonya N. , Brown David A. , Shaikh Saame Raza
Published: November 2023

Cardiac phospholipids, notably cardiolipin, undergo acyl chain remodeling and/or loss of content in diseases such as aging, diabetes and cardiovascular disease. Acyl chain remodeling and loss of content are postulated to mediate mitochondrial dysfunction in these disease states. Surprisingly, less is known about how diet‐induced obesity influences phospholipid acyl chain composition and thereby mitochondrial responses. Here we tested if a high fat diet remodeled murine cardiac phospholipid acyl chain composition and the mitochondrial response. Several mitochondrial endpoints were assayed in response to a high fat diet. We found that mice consuming a high fat diet displayed 0.2–3.3 fold changes in acyl chain remodeling of cardiolipin, phosphatidylcholine, and phosphatidylethanolamine, measured via LC/MS, but no change in total phospholipid mass. Analysis of monolayers constructed using a Langmuir trough from the mitochondrial phospholipids of obese mice revealed an accompanying impairment in the packing properties of the membrane. Although there were alterations in the mitochondrial membrane composition and organization, the high fat diet, relative to the lean controls, had no influence on cardiac mitochondrial supercomplex formation, respiratory enzyme activity, or respiration. These effects were tissue specific however, since complex I to III liver respiratory enzyme activity was diminished with the high fat diet compared to the control diet. These results suggest that diet‐induced obesity leads to modest acyl chain remodeling of mitochondrial phospholipids but does not disrupt mitochondrial supercomplexes, respiratory enzyme activity, or respiration in the heart. Altogether, our findings support an emerging view suggesting that mitochondrial acyl chain remodeling is not a major driver of disrupting mitochondrial function.

Support or Funding Information

Funding supported by NIH R15HL12292201 (SRS and DAB) and NIH R01HL123647 (SRS and DAB).
Analysis of Oct1p/MIPEP Proteolytic Processing of Coenzyme Q Biosynthesis Proteins
Authors: Drahnak Michael J , Veling Mike T , Pagliarini David J
Published: November 2023

Mitoproteases are emerging as key regulators of diverse mitochondrial functions, although their direct substrates are often difficult to discern. Using multi‐omic analyses and machine learning, we have predicted a distinct association between one mitoprotease in Saccharomyces cerevisiae, Oct1p, and coenzyme Q (CoQ) biosynthesis—a pathway essential for mitochondrial respiration. More specifically, we have discovered that Oct1p directly processes the N‐terminus of at least one CoQ‐related biosynthesis enzyme. Mutations to the N‐terminus of this substrate inhibit Oct1p processing in vivo, leading to CoQ deficiency and subsequent respiratory in competency. The objective of this study is to confirm in vitro that Oct1p and its disease‐related human ortholog MIPEP assist in the biosynthesis of CoQ via processing of one or more proteins in the CoQ pathway. Recombinant Oct1p and MIPEP were expressed in Escherichia coli cells and were purified via dual immobilized metal affinity chromatography (IMAC). Protease activity was quantified using Fluorescence Resonance Energy Transfer (FRET) peptides. Activity for both Oct1p and MIPEP was verified using generic control peptides, enabling us to test CoQ‐related proteins as direct substrates. This work reveals the importance of a post‐translational processing event for CoQ biosynthesis, and connects a mitoprotease to a new mitochondrial function.
Single, long molecule PCR for the detection of rare mutations in Mitochondrial DNA
Authors: Li Housaiyin , Annis Sofia , Woods Dori , Tilly Jonathan , Khrapko Konstantin
Published: November 2023

Introduction

Oocytes contain the whole mitochondrial genomes of mammals, and the inheritance pattern of mitochondrial DNA in oocytes do not follow Mendel's rules of heredity. Mitochondrial DNA is maternally inherited in the oocytes, and only mother's mitochondrial genome can be passed to the next generation. The mitochondrial genome is a closed, circular piece of DNA which has a length of about 16kb, and there are hundreds to hundreds of thousands mitochondrial DNA in a single cell. Mitochondrial DNA mutation rate is generally difficult to detect and is often over‐estimated. This research is focusing on developing a sequencing strategy to accurately get mitochondrial DNA mutation rate of single, long molecules from a wild type mouse germline.

Research Objectives

The research goal is to develop an efficient and accurate way to assess the mutation rate of mitochondrial DNA from wild type mouse oocytes.

Methods

We isolated and individually lysed single oocytes from a wild type female mouse. A serial dilution of the DNA sample was made to find its working concentration for generating single, long molecules, and prepared a PCR reaction by using the specific DNA sample concentration. A high‐fidelity DNA polymerase was used to generate 16kb PCR products originating from single template molecules. Sanger sequencing was used to sequence these DNA molecules.

Results

We used single molecule PCR to amplify the mitochondrial DNA from three wild type mouse oocytes and sequenced all the PCR products, yielding a total coverage about 330,392bp. Twenty‐two mitochondrial genomes from three oocytes were sequenced, with an average length of about 15,017bp. Mouse mitochondrial DNA length is 16,299kb, so each of our mitochondrial DNA molecule has an average coverage about 92.13% of the whole mitochondrial DNA genome. Only two mutations were found in two separate mitochondrial genomes, and the resulting mutation rate of mitochondrial DNA in the oocytes was 6.05×10^‐6. This is a very low mutation rate across over 300,000bp.

Conclusion

The mutation rate of mitochondrial DNA in wild type mouse germline is remarkable low in our research by using single, long molecule PCR. This PCR method allows for the accurate detection of mutations in the mouse mitochondrial DNA in oocytes. Our data indicates that, by using single, long molecule PCR combined with Sanger sequencing, we can accurately get informations from a molecule that has the most coverage of its genome. Additionally, we used a high‐fidelity enzyme to serve as DNA polymerase in the PCR reaction, thus preventing the chance of artificial mutations from the beginning of the PCR reaction. With the right procedure of our sequencing strategy, the rare mutations and the mutation rate in single germline cells can be accurately determined.

Support or Funding Information

Ellison Medical Foundation; NIH R37‐AG012279
Fis1 activity in pre‐ and post‐assembly of the yeast mitochondrial fission machinery.
Authors: Harwig Megan Cleland , Hill R. Blake
Published: November 2023

Far from static organelles, mitochondria frequently undergo fusion and fission events to help maintain a healthy mitochondrial network. These processes are vital for a variety of cellular tasks including maintenance of the mitochondrial genome as well as the mitochondrial membrane potential. In yeast, a mitochondrial outer membrane fission protein 1 (Fis1) is essential for mitochondrial fission; however, the precise role of Fis1 has been a matter of debate. Fis1 is highly conserved and interacts with two cytosolic fission proteins, Mdv1 and Dnm1 to possibly recruit them to sites of scission, however, the details of these interactions are not well understood. We recently demonstrated that mutating a key residue (E78) of Fis1 helped demonstrate a post‐recruitment role for Fis1. Here, our analyses with additional point mutations of Fis1 points to a revised model for Fis1 activity in yeast mitochondrial fission.

Support or Funding Information

NIH R01 GM067180