NSF and p97/VCP: similar at first, different at last

AbstractN-Ethylmaleimide sensitive factor (NSF) and p97/valosin-containing protein (VCP) are distantly related members of the ATPases associated with a variety of cellular activities (AAA) family of proteins. While both proteins have been implied in cellular morphology changes involving membrane compartments or vesicles, more recent evidence seems to imply that NSF is primarily involved in the soluble NSF attachment receptor (SNARE)-mediated vesicle fusion by disassembling the SNARE complex whereas p97/VCP is primarily involved in the extraction of membrane proteins. These functional differences are now corroborated by major structural differences based on recent crystallographic and cryo-electron microscopy studies. This review discusses these recent findings

X-ray structure determination at low resolution

Refinement is meaningful even at 4 Å or lower, but with present methodologies it should start from high-resolution crystal structures whenever possible

Mesenchymal phenotype predisposes lung cancer cells to impaired proliferation and redox stress in response to glutaminase inhibition

Recent work has highlighted glutaminase (GLS) as a key player in cancer cell metabolism, providing glutamine-derived carbon and nitrogen to pathways that support proliferation. There is significant interest in targeting GLS for cancer therapy, although the gene is not known to be mutated or amplified in tumors. As a result, identification of tractable markers that predict GLS dependence is needed for translation of GLS inhibitors to the clinic. Herein we validate a small molecule inhibitor of GLS and show that non-small cell lung cancer cells marked by low E-cadherin and high vimentin expression, hallmarks of a mesenchymal phenotype, are particularly sensitive to inhibition of the enzyme. Furthermore, lung cancer cells induced to undergo epithelial to mesenchymal transition (EMT) acquire sensitivity to the GLS inhibitor. Metabolic studies suggest that the mesenchymal cells have a reduced capacity for oxidative phosphorylation and increased susceptibility to oxidative stress, rendering them unable to cope with the perturbations induced by GLS inhibition. These findings elucidate selective metabolic dependencies of mesenchymal lung cancer cells and suggest novel pathways as potential targets in this aggressive cancer type

Dimerization of Recombinant Tobacco Mosaic Virus Movement Protein

The p30 movement protein (MP) is essential for cell-to-cell spread of tobacco mosaic virus in planta. We used anion-exchange chromatography and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to obtain highly purified 30-kDa MP, which migrated as a single band in native PAGE. Analytical ultracentrifugation suggested that the protein was monodisperse and dimeric in the nonionic detergent n-octyl-β-d-glucopyranoside. Circular dichroism (CD) spectroscopy showed that the detergent-solubilized protein contained significant α-helical secondary structure. Proteolysis of the C-tail generated a trypsin-resistant core that was a mixture of primarily monomers and some dimers. We propose that MP dimers are stabilized by electrostatic interactions in the C terminus as well as hydrophobic interactions between putative transmembrane α-helical coiled coils

Crystal Structure of a HyperactiveEscherichia coliGlycerol Kinase Mutant Gly230 → Asp Obtained Using Microfluidic Crystallization Devices

The crystal structure of an Escherichia coli glycerol kinase mutant Gly230 → Asp (GKG230D) was determined to 2.0 Å resolution using a microfluidics based crystallization platform. The crystallization strategy involved a suite of microfluidic devices that characterized the solubility trends of GKG230D, performed nanoliter volume free interface diffusion crystallization experiments, and produced diffraction-quality crystals for in situ data collection. GKG230D displays increased enzymatic activity and decreased allosteric regulation by the glycolytic pathway intermediate fructose 1,6-bisphosphate (FBP) compared to wild-type GK (GKWT). Structural analysis revealed that the decreased allosteric regulation is a result of the altered FBP binding loop conformations in GKG230D that interfere with the wild-type FBP binding site. The altered FBP binding loop conformations in GKG230D are supported through a series of intramolecular loop interactions. The appearance of Asp230 in the FBP binding loops also repositions the wild-type FBP binding residues away from the FBP binding site. Light scattering analysis confirmed GKG230D is a dimer and is resistant to tetramer formation in the presence of FBP, whereas GKWT dimers are converted into putatively inactive tetramers in the presence of FBP. GKG230D also provides the first structural evidence for multiple GK monomer conformations in the presence of glycerol and in the absence of a nucleotide substrate and verifies that glycerol binding is not responsible for locking GK into the closed conformation necessary for GK activity

Crystal Structure of a HyperactiveEscherichia coliGlycerol Kinase Mutant Gly230 → Asp Obtained Using Microfluidic Crystallization Devices

The crystal structure of an Escherichia coli glycerol kinase mutant Gly230 → Asp (GKG230D) was determined to 2.0 Å resolution using a microfluidics based crystallization platform. The crystallization strategy involved a suite of microfluidic devices that characterized the solubility trends of GKG230D, performed nanoliter volume free interface diffusion crystallization experiments, and produced diffraction-quality crystals for in situ data collection. GKG230D displays increased enzymatic activity and decreased allosteric regulation by the glycolytic pathway intermediate fructose 1,6-bisphosphate (FBP) compared to wild-type GK (GKWT). Structural analysis revealed that the decreased allosteric regulation is a result of the altered FBP binding loop conformations in GKG230D that interfere with the wild-type FBP binding site. The altered FBP binding loop conformations in GKG230D are supported through a series of intramolecular loop interactions. The appearance of Asp230 in the FBP binding loops also repositions the wild-type FBP binding residues away from the FBP binding site. Light scattering analysis confirmed GKG230D is a dimer and is resistant to tetramer formation in the presence of FBP, whereas GKWT dimers are converted into putatively inactive tetramers in the presence of FBP. GKG230D also provides the first structural evidence for multiple GK monomer conformations in the presence of glycerol and in the absence of a nucleotide substrate and verifies that glycerol binding is not responsible for locking GK into the closed conformation necessary for GK activity

EMT drives GLS1 dependence in two independent NSCLC models.

<p>(<b>A</b>) A427 cells were transfected with non-targeting (NT) or β-catenin targeting siRNAs and treated with the indicated concentrations of BPTES (left panel). Growth rates were normalized to DMSO treated cells transfected with NT siRNAs. The right panel depicts the effects of the β-catenin siRNAs on growth of DMSO treated cells. <b>(B</b>) Immunoblot of A427 protein extracts collected from cells 72 hrs post transfection. (<b>C</b>) NCI-H358 cells were treated with 25ng/ml TGF-ß for 4 wks. Induction of EMT was confirmed by the loss of E-cadherin and gain of vimentin. EMT was accompanied by an increase in GAC relative to PC protein levels (results representative of 2 independent experiments). Actin levels indicated for protein normalization. (<b>D</b>) NCI-H358 parental and 6wk TGF-ß treated cells were treated in triplicate with BPTES for 72 hrs and cell growth assessed by CTG. Results are representative of 3 independent experiments and are plotted as average growth rates (+/−SD) compared to DMSO treated cells.</p

Metabolic profiling of epithelial and mesenchymal cells and effects of GLS inhibition.

<p>NCI-H358 epithelial (E) and mesenchymal (M) lines were treated with DMSO or 8 µM BPTES for 20 hrs. Cells were either unlabeled or U-¹³C-Glc or U-¹³C-Gln labels were included for the last 4 hrs of drug treatment and levels of central carbon metabolites were measured by LCMS. (A) Inhibition of GLS activity by BPTES. (B) Percentage of citrate pool that is either unlabeled or containing carbon derived from U-¹³C-Gln (DMSO treated cells). (C) TCA metabolite and glutathione pool sizes +/− BPTES. Data was collected in triplicate or quadruplicate from 3 independent experiments (for α-KG and citrate) and depicted as mean levels +/−SD relative to NCI-H358 DMSO treated cells; values were normalized for cell number; <i>P</i> =  *1×10⁻⁵; **4×10⁻⁶; ***9×10⁻⁸ (D) Citrate isotopomer percentages from ¹³C(6)-Glc labeled cells treated +/− BPTES. *<i>P</i> = 0.001, **<i>P</i> = 0.002 comparing % citrate m+2 in DMSO or BPTES treated E vs M cells, respectively. (E) Alterations in relative ratios of DHAP and 3PG levels in EvsM cells +/−BPTES. <i>P</i> values for comparison of metabolite levels +/−BPTES: *0.1, **0.57, ^#0.04, ^##0.07. Results representative of 2 independent experiments.</p