Hydrolysis Rates of Different Small Interfering RNAs (siRNAs) by the RNA Silencing Promoter Complex,C3PO,Determines Their Regulation by Phospholipase Cβ

C3PO plays a key role in promoting RNA-induced gene silencing. C3PO consists of two subunits of the endonuclease translin-associated factor X (TRAX) and six subunits of the nucleotide-binding protein translin. We have found that TRAX binds strongly to phospholipase Cβ (PLCβ), which transmits G protein signals from many hormones and sensory inputs. The association between PLCβ and TRAX is thought to underlie the ability of PLCβ to reverse gene silencing by small interfering RNAs. However, this reversal only occurs for some genes (e.g. GAPDH and LDH) but not others (e.g. Hsp90 and cyclophilin A). To understand this specificity, we carried out studies using fluorescence-based methods. In cells, we find that PLCβ, TRAX, and their complexes are identically distributed through the cytosol suggesting that selectivity is not due to large scale sequestration of either the free or complexed proteins. Using purified proteins, we find that PLCβ binds ∼5-fold more weakly to translin than to TRAX but ∼2-fold more strongly to C3PO. PLCβ does not alter TRAX-translin assembly to C3PO, and brightness studies suggest one PLCβ binds to one C3PO octamer without a change in the number of TRAX/translin molecules suggesting that PLCβ binds to an external site. Functionally, we find that C3PO hydrolyzes siRNA(GAPDH) at a faster rate than siRNA(Hsp90). However, when PLCβ is bound to C3PO, the hydrolysis rate of siRNA(GAPDH) becomes comparable with siRNA(Hsp90). Our results show that the selectivity of PLCβ toward certain genes lies in the rate at which the RNA is hydrolyzed by C3PO.     

Studies on the Mechanism of Electron Bifurcation Catalyzed by Electron Transferring Flavoprotein (Etf) and Butyryl-CoA Dehydrogenase (Bcd) of Acidaminococcus fermentans

Electron bifurcation is a fundamental strategy of energy coupling originally discovered in the Q-cycle of many organisms. Recently a flavin-based electron bifurcation has been detected in anaerobes, first in clostridia and later in acetogens and methanogens. It enables anaerobic bacteria and archaea to reduce the low-potential [4Fe-4S] clusters of ferredoxin, which increases the efficiency of the substrate level and electron transport phosphorylations. Here we characterize the bifurcating electron transferring flavoprotein (Etf_Af) and butyryl-CoA dehydrogenase (Bcd_Af) of Acidaminococcus fermentans, which couple the exergonic reduction of crotonyl-CoA to butyryl-CoA to the endergonic reduction of ferredoxin both with NADH. Etf_Af contains one FAD (α-FAD) in subunit α and a second FAD (β-FAD) in subunit β. The distance between the two isoalloxazine rings is 18 Å. The Etf_Af-NAD⁺ complex structure revealed β-FAD as acceptor of the hydride of NADH. The formed β-FADH⁻ is considered as the bifurcating electron donor. As a result of a domain movement, α-FAD is able to approach β-FADH⁻ by about 4 Å and to take up one electron yielding a stable anionic semiquinone, α-FAD^⨪, which donates this electron further to Dh-FAD of Bcd_Af after a second domain movement. The remaining non-stabilized neutral semiquinone, β-FADH^•, immediately reduces ferredoxin. Repetition of this process affords a second reduced ferredoxin and Dh-FADH⁻ that converts crotonyl-CoA to butyryl-CoA.     

Degradation of Phycobilisomes in Synechocystis sp. PCC6803: EVIDENCE FOR ESSENTIAL FORMATION OF AN NblA1/NblA2 HETERODIMER AND ITS CODEGRADATION BY A Clp PROTEASE COMPLEX

When cyanobacteria acclimate to nitrogen deficiency, they degrade their large (3–5-MDa), light-harvesting complexes, the phycobilisomes. This massive, yet specific, intracellular degradation of the pigmented phycobiliproteins causes a color change of cyanobacterial cultures from blue-green to yellow-green, a process referred to as chlorosis or bleaching. Phycobilisome degradation is induced by expression of the nblA gene, which encodes a protein of ∼7 kDa. NblA most likely acts as an adaptor protein that guides a Clp protease to the phycobiliproteins, thereby initiating the degradation process. Most cyanobacteria and red algae possess just one nblA-homologous gene. As an exception, the widely used “model organism” Synechocystis sp. PCC6803 expresses two such genes, nblA1₆₈₀₃ and nblA2₆₈₀₃, both of whose products are required for phycobilisome degradation. Here, we demonstrate that the two NblA proteins heterodimerize in vitro and in vivo using pull-down assays and a Förster energy-transfer approach, respectively. We further show that the NblA proteins form a ternary complex with ClpC (the HSP100 chaperone partner of Clp proteases) and phycobiliproteins in vitro. This complex is susceptible to ATP-dependent degradation by a Clp protease, a finding that supports a proposed mechanism of the degradation process. Expression of the single nblA gene encoded by the genome of the N₂-fixing, filamentous cyanobacterium Nostoc sp. PCC7120 in the nblA1/nblA2 mutant of Synechocystis sp. PCC6803 induced phycobilisome degradation, suggesting that the function of the NblA heterodimer of Synechocystis sp. PCC6803 is combined in the homodimeric protein of Nostoc sp. PCC7120.     

Direct Determination of Multiple Ligand Interactions with the Extracellular Domain of the Calcium-sensing Receptor

Numerous in vivo functional studies have indicated that the dimeric extracellular domain (ECD) of the CaSR plays a crucial role in regulating Ca²⁺ homeostasis by sensing Ca²⁺ and l-Phe. However, direct interaction of Ca²⁺ and Phe with the ECD of the receptor and the resultant impact on its structure and associated conformational changes have been hampered by the large size of the ECD, its high degree of glycosylation, and the lack of biophysical methods to monitor weak interactions in solution. In the present study, we purified the glycosylated extracellular domain of calcium-sensing receptor (CaSR) (ECD) (residues 20–612), containing either complex or high mannose N-glycan structures depending on the host cell line employed for recombinant expression. Both glycosylated forms of the CaSR ECD were purified as dimers and exhibit similar secondary structures with ∼50% α-helix, ∼20% β-sheet content, and a well buried Trp environment. Using various spectroscopic methods, we have shown that both protein variants bind Ca²⁺ with a K_d of 3.0–5.0 mm. The local conformational changes of the proteins induced by their interactions with Ca²⁺ were visualized by NMR with specific ¹⁵N Phe-labeled forms of the ECD. Saturation transfer difference NMR approaches demonstrated for the first time a direct interaction between the CaSR ECD and l-Phe. We further demonstrated that l-Phe increases the binding affinity of the CaSR ECD for Ca²⁺. Our findings provide new insights into the mechanisms by which Ca²⁺ and amino acids regulate the CaSR and may pave the way for exploration of the structural properties of CaSR and other members of family C of the GPCR superfamily.     

Phospholamban C-terminal Residues Are Critical Determinants of the Structure and Function of the Calcium ATPase Regulatory Complex

To determine the structural and regulatory role of the C-terminal residues of phospholamban (PLB) in the membranes of living cells, we fused fluorescent protein tags to PLB and sarco/endoplasmic reticulum calcium ATPase (SERCA). Alanine substitution of PLB C-terminal residues significantly altered fluorescence resonance energy transfer (FRET) from PLB to PLB and SERCA to PLB, suggesting a change in quaternary conformation of PLB pentamer and SERCA-PLB regulatory complex. Val to Ala substitution at position 49 (V49A) had particularly large effects on PLB pentamer structure and PLB-SERCA regulatory complex conformation, increasing and decreasing probe separation distance, respectively. We also quantified a decrease in oligomerization affinity, an increase in binding affinity of V49A-PLB for SERCA, and a gain of inhibitory function as quantified by calcium-dependent ATPase activity. Notably, deletion of only a few C-terminal residues resulted in significant loss of PLB membrane anchoring and mislocalization to the cytoplasm and nucleus. C-terminal truncations also resulted in progressive loss of PLB-PLB FRET due to a decrease in the apparent affinity of PLB oligomerization. We quantified a similar decrease in the binding affinity of truncated PLB for SERCA and loss of inhibitory potency. However, despite decreased SERCA-PLB binding, intermolecular FRET for Val⁴⁹-stop (V49X) truncation mutant was paradoxically increased as a result of an 11.3-Å decrease in the distance between donor and acceptor fluorophores. We conclude that PLB C-terminal residues are critical for localization, oligomerization, and regulatory function. In particular, the PLB C terminus is an important determinant of the quaternary structure of the SERCA regulatory complex.     

Syntenin-1 and Ezrin Proteins Link Activated Leukocyte Cell Adhesion Molecule to the Actin Cytoskeleton

Activated leukocyte cell adhesion molecule (ALCAM) is a type I transmembrane protein member of the immunoglobulin superfamily of cell adhesion molecules. Involved in important pathophysiological processes such as the immune response, cancer metastasis, and neuronal development, ALCAM undergoes both homotypic interactions with other ALCAM molecules and heterotypic interactions with the surface receptor CD6 expressed at the T cell surface. Despite biochemical and biophysical evidence of a dynamic association between ALCAM and the actin cytoskeleton, no detailed information is available about how this association occurs at the molecular level. Here, we exploit a combination of complementary microscopy techniques, including FRET detected by fluorescence lifetime imaging microscopy and single-cell force spectroscopy, and we demonstrate the existence of a preformed ligand-independent supramolecular complex where ALCAM stably interacts with actin by binding to syntenin-1 and ezrin. Interaction with the ligand CD6 further enhances these multiple interactions. Altogether, our results propose a novel biophysical framework to understand the stabilizing role of the ALCAM supramolecular complex engaged to CD6 during dendritic cell-T cell interactions and provide novel information on the molecular players involved in the formation and signaling of the immunological synapse at the dendritic cell side.     

Biochemical Characterization of Pathogenic Mutations in Human Mitochondrial Methionyl-tRNA Formyltransferase

N-Formylation of initiator methionyl-tRNA (Met-tRNA^Met) by methionyl-tRNA formyltransferase (MTF) is important for translation initiation in bacteria, mitochondria, and chloroplasts. Unlike all other translation systems, the metazoan mitochondrial system is unique in using a single methionine tRNA (tRNA^Met) for both initiation and elongation. A portion of Met-tRNA^Met is formylated for initiation, whereas the remainder is used for elongation. Recently, we showed that compound heterozygous mutations within the nuclear gene encoding human mitochondrial MTF (mt-MTF) significantly reduced mitochondrial translation efficiency, leading to combined oxidative phosphorylation deficiency and Leigh syndrome in two unrelated patients. Patient P1 has a stop codon mutation in one of the MTF genes and an S209L mutation in the other MTF gene. P2 has a S125L mutation in one of the MTF genes and the same S209L mutation as P1 in the other MTF gene. Here, we have investigated the effect of mutations at Ser-125 and Ser-209 on activities of human mt-MTF and of the corresponding mutations, Ala-89 or Ala-172, respectively, on activities of Escherichia coli MTF. The S125L mutant has 653-fold lower activity, whereas the S209L mutant has 36-fold lower activity. Thus, both patients depend upon residual activity of the S209L mutant to support low levels of mitochondrial protein synthesis. We discuss the implications of these and other results for whether the effect of the S209L mutation on mitochondrial translational efficiency is due to reduced activity of the mutant mt-MTF and/or reduced levels of the mutant mt-MTF.     

Lipoprotein subclasses and endogenous sex hormones in women at midlife

The objective of this work was to evaluate the associations between levels of endogenous sex hormones in women at midlife and lipoprotein subclasses. One hundred and twenty women (68 late peri-/postmenopausal and 52 pre-/early perimenopausal) from the Study of Women’s Health Across the Nation (Pittsburgh site) were included. Lipoprotein subclasses were quantified using NMR spectroscopy. Participants (57.5% White and 42.5% Black) were 50.4 ± 1.9 years old. Adjusting for age, race, cycle day of blood draw, BMI, physical activity, and alcohol consumption, a negative correlation was found between estradiol (E2) and medium-small LDL particle (LDL-P) concentration (ρ = −0.19, P = 0.04). Further, E2 was positively correlated with HDL particle (HDL-P) size (ρ = 0.22, P = 0.02). For sex hormone binding globulin (SHBG), independent negative correlation was found with total small LDL-P concentration. SHBG was also positively correlated with LDL-P and HDL-P sizes (P < 0.05 for all). For free androgen index (FAI), positive correlations were found with concentrations of total VLDL particles, total LDL-Ps, and total small LDL-Ps. Additionally, FAI was negatively correlated with large HDL-P concentration, and HDL-P and LDL-P sizes (P < 0.05 for all). Lower levels of E2 and SHBG, and higher levels of FAI were associated with a more atherogenic profile of lipoprotein subclasses. Sex hormone levels at midlife may increase women’s risk of coronary heart disease.     

NP-Sticky: A Web Server for Optimizing DNA Ligation with Non-Palindromic Sticky Ends

High-efficiency DNA ligation is vital for many molecular biology experiments, and it is best achieved using reactants with non-palindromic sticky ends to maximize specificity. However, optimizing such multi-parametric ligation reactions often involves extensive trial and error. We have developed a freely available Web-based ligation calculator, NP-Sticky (http://sarkarlab.umn.edu/npsticky/), that predicts product distribution for given reactant concentrations, thus enabling straightforward computational optimization of these reactions. Built-in schemes include two-piece and three-piece linear ligation, as well as insert-vector circular ligation. The only parameters needed for the underlying thermodynamic model are the free energies of ligation for each sticky end, which can be estimated by the calculator from the overhang sequences or provided by the user from direct experimental measurement. Free energies of sticky-end mismatches are also calculated for determining the extent of byproduct formation. This ligation calculator allows rapid identification of the optimal conditions for maximizing incorporation, efficiency, and/or accuracy, based on specific needs.     

Female polyandry and size‐assortative mating in isolated local populations of the Japanese common toad Bufo japonicus

In anurans, female polyandry under male harassment is distributed across taxa because of external aquatic fertilization. According to the sexual selection theory, male–male competition for access to females is affected by the operational sex ratio (OSR) and population density. The Japanese common toad, Bufo japonicus, is widespread in mainland Japan, and like the European common toad, B. bufo, it engages in explosive breeding. In this study, we observed the breeding behaviour of B. japonicus in isolated local populations for over four years in two breeding ponds with different population sizes and densities: large‐low (L‐pond) and small‐high (S‐pond). We analysed the relative polyandry ratio in egg clutches laid by females and estimated the size‐assortative mating pattern to be an indicator of male–male competition in the two ponds. Both ponds tended to exhibit a size‐assortative mating pattern; however, the frequency of polyandry was different in the two ponds (L‐pond = 20% and S‐pond = 90%). Our results showed that polyandry could occur without multiple amplexus with a high population density, i.e. eggs were often fertilized by free‐swimming sperm in the small shallow pond. We propose that high female polyandry ratios without continuous male harassment are generated because of a male‐biased OSR and a high population density in the small pond. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 113 , 236–242.