Distinctive roles of receptor‐interacting protein kinases 1 and 3 in caspase‐independent cell death of L929

Upon tumour necrosis factor alpha (TNFα) stimulation, cells respond actively by way of cell survival, apoptosis or programmed necrosis. The receptor‐interacting proteins 1 (RIP1) and 3 (RIP3) are responsible for TNFα‐mediated programmed necrosis. To delineate the differential contributions of RIP3 and RIP1 to programmed necrosis, L929 cells were stimulated with TNFα, carbobenzoxy‐valyl‐alanyl‐aspartyl‐[O‐methyl]‐fluoromethylketone (zVAD) or zVAD along with TNFα following RNA interference against RIP1 and RIP3, respectively. RIP1 silencing did not protect cells from TNFα‐mediated cell death, while RIP3 down‐regulation made them refractory to TNFα. The heat shock protein 90 inhibitor geldanamycin (GA) down‐regulated both RIP1 and RIP3 expression, which rendered cells resistant to zVAD/TNFα‐mediated cell death but not to TNFα‐mediated cell death alone. Therefore, the protective effect of GA on zVAD/TNFα‐stimulated necrosis might be attributed to RIP3, not RIP1, down‐regulation. Pretreatment of L929 cells with rapamycin mitigated zVAD‐mediated cell death, while the autophagy inhibitor chloroquine did not affect necrotic cell death. Meanwhile, necrotic cell death by zVAD and TNFα was caused by reactive oxygen species generation and effectively diminished by lipid‐soluble butylated hydroxyanisole. Taken together, the results indicate that RIP1 and RIP3 can independently mediate death signals being transduced by two different death stimuli, zVAD and TNFα. Copyright © 2013 John Wiley & Sons, Ltd.     

2-(Trimethylammonium) Ethyl (R)-3-Methoxy-3-oxo-2-Stearamidopropyl Phosphate Suppresses Osteoclast Maturation and Bone Resorption by Targeting Macrophage-Colony Stimulating Factor Signaling

2-(Trimethylammonium) ethyl (R)-3-methoxy-3-oxo-2-stearamidopropyl phosphate [(R)-TEMOSPho], a derivative of an organic chemical identified from a natural product library, promotes highly efficient megakaryopoiesis. Here, we show that (R)-TEMOSPho blocks osteoclast maturation from progenitor cells of hematopoietic origin, as well as blocking the resorptive function of mature osteoclasts. The inhibitory effect of (R)-TEMOSPho on osteoclasts was due to a disruption of the actin cytoskeleton, resulting from impaired downstream signaling of c-Fms, a receptor for macrophage-colony stimulating factor linked to c-Cbl, phosphoinositol-3-kinase (PI3K), Vav3, and Rac1. In addition, (R)-TEMOSPho blocked inflammation-induced bone destruction by reducing the numbers of osteoclasts produced in mice. Thus, (R)-TEMOSPho may represent a promising new class of antiresorptive drugs for the treatment of bone loss associated with increased osteoclast maturation and activity.     

ULK2 Ser 1027 Phosphorylation by PKA Regulates Its Nuclear Localization Occurring through Karyopherin Beta 2 Recognition of a PY-NLS Motif

Uncoordinated 51-like kinase 2 (ULK2), a member of the serine/threonine kinase family, plays an essential role in the regulation of autophagy in mammalian cells. Given the role of autophagy in normal cellular homeostasis and in multiple diseases, improved mechanistic insight into this process may result in the development of novel therapeutic approaches. Here, we present evidence that ULK2 associates with karyopherin beta 2 (Kapβ2) for its transportation into the nucleus. We identify a potential PY-NLS motif (⁷⁷⁴gpgfgssppGaeaapslRyvPY⁷⁹⁵) in the S/P space domain of ULK2, which is similar to the consensus PY-NLS motif (R/K/H)X _2–5PY. Using a pull-down approach, we observe that ULK2 interacts physically with Kapβ2 both in vitro and in vivo. Confocal microscopy confirmed the co-localization of ULK2 and Kapβ2. Localization of ULK2 to the nuclear region was disrupted by mutations in the putative Kapβ2-binding motif (P794A). Furthermore, in transient transfection assays, the presence of the Kapβ2 binding site mutant (the cytoplasmic localization form) was associated with a substantial increase in autophagy activity (but a decrease in the in vitro serine-phosphorylation) compared with the wild type ULK2. Mutational analysis showed that the phosphorylation on the Ser1027 residue of ULK2 by Protein Kinase A (PKA) is the regulatory point for its functional dissociation from Atg13 and FIP 200, nuclear localization, and autophagy. Taken together, our observations indicate that Kapβ2 interacts with ULK2 through ULK2’s putative PY-NLS motif, and facilitates transport from the cytoplasm to the nucleus, depending on its Ser1027 residue phosphorylation by PKA, thereby reducing autophagic activity.     

Fetal mouse selenophosphate synthetase 2 (SPS2): biological activities of mutant forms in Escherichia coli.

A novel gene, sps2, detected in mouse embryo at the early stages of development has been identified as an analog of the E. coli selenophosphate synthetase gene. Unlike the E. coli enzyme, the presence of selenocysteine in the mouse enzyme is indicated by a TGA codon in the open reading frame of the cDNA. Using an N-FLAG monoclonal antibody, it was shown that the full length N-FLAG-sps2 gene product was expressed in COS-7 cells. To investigate the biological activity of the sps2 gene product in vivo, the mutated sps2 gene, which contains cysteine in the place of the TGA encoded selenocysteine in the wild type, was expressed in the E. coli selD deficient mutant, MB08. Like the E. coli wild type selD gene, the mutant sps2 gene complemented the selD mutation. However, replacement of Cys with either Ala, Ser, or Thr resulted in a loss of ability to complement the selD mutation. The SPS2-CYS protein expressed in E. coli was purified and its catalytic activity was determined. The Km value for ATP was 0.75 mM and Vmax was 9.23 nmole/min/mg protein. These results confirm that the mouse embryonic sps2 gene encodes an eukaryotic selenophosphate synthetase, and that availability of selenophosphate as a selenium donor compound is widespread.     

Theoretical calculation of the sugar concentrations during enzymatic production of fructooligosaccharides

Summary In a batch production of fructooligosaccharides from sucrose, the concentrations of residual sucrose, glucose and fructooligosaccharides at a given reaction time(t) and initial sucrose concentration(S₀) were theoretically calculated by the following correlation equations: Glucose(t) = 0.0653 S₀ × ln(t); Fructooligosaccharides(t) = 0.1636 S₀ × ln(t); Sucrose(t)=S₀ - Glucose(t) + FOS(t).     

Single-molecule fluorescence measurements reveal the reaction mechanisms of the core-RISC,composed of human Argonaute 2 and a guide RNA

In eukaryotes, small RNAs play important roles in both gene regulation and resistance to viral infection. Argonaute proteins have been identified as a key component of the effector complexes of various RNA-silencing pathways, but the mechanistic roles of Argonaute proteins in these pathways are not clearly understood. To address this question, we performed single-molecule fluorescence experiments using an RNA-induced silencing complex (core-RISC) composed of a small RNA and human Argonaute 2. We found that target binding of core-RISC starts at the seed region of the guide RNA. After target binding, four distinct reactions followed: target cleavage, transient binding, stable binding, and Argonaute unloading. Target cleavage required extensive sequence complementarity and accelerated core-RISC dissociation for recycling. In contrast, the stable binding of core-RISC to target RNAs required seed-match only, suggesting a potential explanation for the seed-match rule of microRNA (miRNA) target selection. [BMB Reports 2015; 48(12): 643-644]     

ComC is required for the processing and translocation of ComGC, a pilin-like competence protein of Bacillus subtilis

ComGC is a cell surface-localized protein required for DNA binding during transformation in Bacillus subtilis. It resembles type IV prepilins in its N-terminal domain, particularly in the amino acid sequence surrounding the processing cleavage sites of these proteins. ComC is another protein required for DNA binding, which resembles the processing proteases that cleave type IV prepilins. We show here that ComGC is processed in competent cells and that this processing requires ComC. We also demonstrate that the PilD protein of Neisseria gonorrhoeae, a ComC homologue, can process ComGC in Escherichia coli, and that the ComC protein itself is the only B. subtilis protein needed to accomplish cleavage of ComGC in the latter organism. Based on NaOH-solubility studies, we have shown that in the absence of ComC, but in the presence of all other competence proteins, B. subtilis is incapable of correctly translocating ComGC to the outer face of the cell membrane. Finally, we show that ComGC can be cross-linked to yield a form with higher molecular mass, possibly a dimer, and present evidence suggesting that formation of the higher mass complex takes place in the membrane, prior to translocation. Formation of this complex does not require ComC or any of the comG products, other than ComGC itself.     

Surface Plasmon Resonance Imaging-Based Protein Array Chip System for Monitoring a Hexahistidine-Tagged Protein during Expression and Purification

A surface plasmon resonance imaging-based Ni²⁺-iminodiacetic acid-coated gold chip system was developed to enable specific detection of a hexahistidine-tagged recombinant protein in crude extracts or in column chromatography fractions. This system is especially advantageous for high-throughput analysis of multiple proteins.