Macromolecular Interactions of Lipoprotein Lipase (LPL).

Lipoprotein lipase (LPL) is a critical enzyme in humans that provides fuel to peripheral tissues. LPL hydrolyzes triglycerides from the cores of lipoproteins that are circulating in plasma and interacts with receptors to mediate lipoprotein uptake, thus directing lipid distribution via catalytic and non-catalytic functions. Functional losses in LPL or any of its myriad of regulators alter lipid homeostasis and potentially affect the risk of developing cardiovascular disease-either increasing or decreasing the risk depending on the mutated protein.

Isocitrate Dehydrogenase Mutations in Cancer: Mechanisms of Transformation and Metabolic Liability.

Isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) are metabolic enzymes that interconvert isocitrate and 2-oxoglutarate (2OG). Gain-of-function mutations in and occur in a number of cancers, including acute myeloid leukemia, glioma, cholangiocarcinoma, and chondrosarcoma. These mutations cripple the wild-type activity of IDH and cause the enzymes to catalyze a partial reverse reaction in which 2OG is reduced but not carboxylated, resulting in production of the ()-enantiomer of 2-hydroxyglutarate (()-2HG).

Structure of dimeric lipoprotein lipase reveals a pore adjacent to the active site.

Lipoprotein lipase (LPL) hydrolyzes triglycerides from circulating lipoproteins, releasing free fatty acids. Active LPL is needed to prevent hypertriglyceridemia, which is a risk factor for cardiovascular disease (CVD). Using cryogenic electron microscopy (cryoEM), we determined the structure of an active LPL dimer at 3.

Combined action of albumin and heparin regulates lipoprotein lipase oligomerization, stability, and ligand interactions.

Lipoprotein lipase (LPL), a crucial enzyme in the intravascular hydrolysis of triglyceride-rich lipoproteins, is a potential drug target for the treatment of hypertriglyceridemia. The activity and stability of LPL are influenced by a complex ligand network. Previous studies performed in dilute solutions suggest that LPL can appear in various oligomeric states.

Structure of Dimeric Lipoprotein Lipase Reveals a Pore for Hydrolysis of Acyl Chains.

Lipoprotein lipase (LPL) hydrolyzes triglycerides from circulating lipoproteins, releasing free fatty acids. Active LPL is needed to prevent hypertriglyceridemia, which is a risk factor for cardiovascular disease (CVD). Using cryogenic electron microscopy (cryoEM), we determined the structure of an active LPL dimer at 3.

Tides regulate the flow and density of Antarctic Bottom Water from the western Ross Sea.

Antarctic Bottom Water (AABW) stores heat and gases over decades to centuries after contact with the atmosphere during formation on the Antarctic shelf and subsequent flow into the global deep ocean. Dense water from the western Ross Sea, a primary source of AABW, shows changes in water properties and volume over the last few decades. Here we show, using multiple years of moored observations, that the density and speed of the outflow are consistent with a release from the Drygalski Trough controlled by the density in Terra Nova Bay (the "accelerator") and the tidal mixing (the "brake").

(R)-2-Hydroxyglutarate Inhibits KDM5 Histone Lysine Demethylases to Drive Transformation in IDH-Mutant Cancers.

Unlabelled: Oncogenic mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 occur in a wide range of cancers, including acute myeloid leukemia (AML) and glioma. Mutant IDH enzymes convert 2-oxoglutarate (2OG) to (R)-2-hydroxyglutarate [(R)-2HG], an oncometabolite that is hypothesized to promote cellular transformation by dysregulating 2OG-dependent enzymes. The only (R)-2HG target that has been convincingly shown to contribute to transformation by mutant IDH is the myeloid tumor suppressor TET2.

Comparison of angiopoietin-like protein 3 and 4 reveals structural and mechanistic similarities.

Elevated plasma triglycerides are a risk factor for coronary artery disease, which is the leading cause of death worldwide. Lipoprotein lipase (LPL) reduces triglycerides in the blood by hydrolyzing them from triglyceride-rich lipoproteins to release free fatty acids. LPL activity is regulated in a nutritionally responsive manner by macromolecular inhibitors including angiopoietin-like proteins 3 and 4 (ANGPTL3 and ANGPTL4).

The structure of helical lipoprotein lipase reveals an unexpected twist in lipase storage.

Lipases are enzymes necessary for the proper distribution and utilization of lipids in the human body. Lipoprotein lipase (LPL) is active in capillaries, where it plays a crucial role in preventing dyslipidemia by hydrolyzing triglycerides from packaged lipoproteins. Thirty years ago, the existence of a condensed and inactive LPL oligomer was proposed.

Protecting activity of desiccated enzymes.

Protein-based biological drugs and many industrial enzymes are unstable, making them prohibitively expensive. Some can be stabilized by formulation with excipients, but most still require low temperature storage. In search of new, more robust excipients, we turned to the tardigrade, a microscopic animal that synthesizes cytosolic abundant heat soluble (CAHS) proteins to protect its cellular components during desiccation.

Nucleation of Multiple Buckled Structures in Intertwined DNA Double Helices.

We study the statistical-mechanical properties of intertwined double-helical DNAs (DNA braids). In magnetic tweezers experiments, we find that torsionally stressed stretched braids supercoil via an abrupt buckling transition, which is associated with the nucleation of a braid end loop, and that the buckled braid is characterized by a proliferation of multiple domains. Differences between the mechanics of DNA braids and supercoiled single DNAs can be understood as an effect of the increased bulkiness in the structure of the former.

Single-Molecule Magnetic Tweezer Analysis of Topoisomerases.

Magnetic tweezers (MT) provide a powerful single-molecule approach to study the mechanism of topoisomerases, giving the experimenter the ability to change and read out DNA topology in real time. By using diverse DNA substrates, one can study different aspects of topoisomerase function and arrive at a better mechanistic understanding of these fascinating enzymes. Here we describe methods for the creation of three different DNA substrates used in MT experiments with topoisomerases: double-stranded DNA (dsDNA) tethers, "braided" (intertwined or catenated) DNA tether pairs, and dsDNA tethers with single-stranded DNA (ssDNA) regions.

An orthogonal single-molecule experiment reveals multiple-attempt dynamics of type IA topoisomerases.

Topoisomerases are enzymes that are involved in maintaining the topological state of cellular DNA. Their dynamic characteristics remain poorly understood despite numerous structural, biophysical and biochemical studies. Recent single-molecule experiments revealed that an important feature of the type IA topoisomerase mechanism is the presence of pauses between relaxation events.

qPCR for second year undergraduates: A short, structured inquiry to illustrate differential gene expression.

We describe a structured inquiry laboratory exercise that examines transcriptional regulation of the NOS2 gene under conditions that simulate the inflammatory response in macrophages. Using quantitative PCR and the comparative CT method, students are able determine whether transcriptional activation of NOS2 occurs and to what degree. The exercise is aimed at second year undergraduates who possess basic knowledge of gene expression events.

Niemann-pick C1 is essential for ebolavirus replication and pathogenesis in vivo.

Unlabelled: Recent work demonstrated that the Niemann-Pick C1 (NPC1) protein is an essential entry receptor for filoviruses. While previous studies focused on filovirus entry requirements of NPC1 in vitro, its roles in filovirus replication and pathogenesis in vivo remain unclear. Here, we evaluated the importance of NPC1, and its partner in cholesterol transport, NPC2, by using a mouse model of Ebolavirus (EBOV) disease.

Inquiry-based learning: inflammation as a model to teach molecular techniques for assessing gene expression.

This laboratory module simulates the process used by working scientists to ask and answer a question of biological interest. Instructors facilitate acquisition of knowledge using a comprehensive, inquiry-based approach in which students learn theory, hypothesis development, experimental design, and data interpretation and presentation. Using inflammation in macrophages as a model system, students perform a series of molecular biology techniques to address the biological question: "Does stimulus 'X' induce inflammation?" To ask this question, macrophage cells are treated with putative inflammatory mediators and then assayed for evidence of inflammatory response.

Bacterial topoisomerase I and topoisomerase III relax supercoiled DNA via distinct pathways.

Escherichia coli topoisomerases I and III (Topo I and Topo III) relax negatively supercoiled DNA and also catenate/decatenate DNA molecules containing single-stranded DNA regions. Although these enzymes share the same mechanism of action and have similar structures, they participate in different cellular processes. In bulk experiments Topo I is more efficient at DNA relaxation, whereas Topo III is more efficient at catenation/decatenation, probably reflecting their differing cellular roles.

CTP:phosphocholine cytidylyltransferase alpha is required for B-cell proliferation and class switch recombination.

CTP:phosphocholine cytidylyltransferase (CCT) is a key rate-controlling enzyme in the biosynthetic pathway leading to the principle membrane phospholipid, phosphatidylcholine. CCTalpha is the predominant isoform expressed in mammalian cells. To investigate the role of CCTalpha in the development and function of B-lymphocytes, mice with B-lymphocytes that selectively lacked CCTalpha were derived using the CD19-driven Cre/loxP system.

Evidence that marginal zone B cells possess an enhanced secretory apparatus and exhibit superior secretory activity.

Marginal zone B (MZB) cells are the first splenic B cells to initiate Ab secretion against polysaccharide-encapsulated Ags in vivo. This swift MZB cell response can be reproduced in vitro as LPS treatment induces Ab secretion in as little as 12 h. Conversely, in vitro LPS treatment of splenic follicular B (FOB) cells results in Ab secretion after 2-3 days.

A role for the unfolded protein response in optimizing antibody secretion.

Terminal differentiation of B lymphocytes into antibody(Ab)-secreting plasma cells is marked by a sharp rise in immunoglobulin (Ig) biosynthesis that increases demand on the protein folding capacity of the endoplasmic reticulum (ER). The unfolded protein response pathway (UPR) allows cells to respond to challenging conditions within the ER, in part by the activities of the XBP1 and ATF6alpha/beta transcription factors. The UPR is activated in differentiating B cells, and XBP1 is required for the generation of Ab-secreting plasma cells.

Stressed-out B cells? Plasma-cell differentiation and the unfolded protein response.

Plasma cells operate as factories where large quantities of Ig heavy and light chains are made and assembled into functional antibodies. The finished products are shipped out with impressive efficiency. A major component of the machinery necessary for high-rate antibody secretion is an elaborate network of endoplasmic reticulum (ER), the site of antibody biosynthesis.

Modulation of CRP-dependent transcription at the Escherichia coli acsP2 promoter by nucleoprotein complexes: anti-activation by the nucleoid proteins FIS and IHF.

acs encodes acetyl-coenzyme A synthetase, a high-affinity enzyme that allows cells to scavenge for acetate during carbon starvation. CRP activates acs transcription by binding tandem DNA sites located upstream of the major promoter, acsP2. Here, we used electrophoretic mobility shift assays and DNase I footprint analyses to demonstrate that the nucleoid proteins FIS and IHF each bind multiple sites within the acs regulatory region, that FIS competes successfully with CRP for binding to their overlapping and neighbouring sites and that IHF binds independently of either FIS or CRP.