Competing Activities of Heterotrimeric G Proteins in Drosophila Wing Maturation

Drosophila genome encodes six alpha-subunits of heterotrimeric G proteins. The Gαs alpha-subunit is involved in the post-eclosion wing maturation, which consists of the epithelial-mesenchymal transition and cell death, accompanied by unfolding of the pupal wing into the firm adult flight organ. Here we show that another alpha-subunit Gαo can specifically antagonize the Gαs activities by competing for the Gβ13F/Gγ1 subunits of the heterotrimeric Gs protein complex. Loss of Gβ13F, Gγ1, or Gαs, but not any other G protein subunit, results in prevention of post-eclosion cell death and failure of the wing expansion. However, cell death prevention alone is not sufficient to induce the expansion defect, suggesting that the failure of epithelial-mesenchymal transition is key to the folded wing phenotypes. Overactivation of Gαs with cholera toxin mimics expression of constitutively activated Gαs and promotes wing blistering due to precocious cell death. In contrast, co-overexpression of Gβ13F and Gγ1 does not produce wing blistering, revealing the passive role of the Gβγ in the Gαs-mediated activation of apoptosis, but hinting at the possible function of Gβγ in the epithelial-mesenchymal transition. Our results provide a comprehensive functional analysis of the heterotrimeric G protein proteome in the late stages of Drosophila wing development.     

Aliphatic ionenes as gene delivery agents: elucidation of structure-function relationship through modification of charge density and polymer length

Polycation-based gene delivery agents are generally polydisperse populations whose properties are averaged among the different molecular weight species. Therefore, to understand the physicochemical properties of polycations and their relationships to cellular gene transfer, one needs to control the molecular weight of the polymer as well as its cationic charge density. To investigate the structure-function correlation of polycations with respect to the degree of polymerization (DP) and charge density, a series of model materials based on aliphatic ionenes was synthesized and fractionated into distinct molecular weight fractions with DP range from 14 to 32. The aliphatic ionene fractions and their polyelectrolyte complexes (PEC) with DNA were studied using physicochemical and biological methods. Ionene polymers were shown to possess low cytotoxicity (minimal viability of the P388D1 murine macrophage cells 80%). DP and charge density of the ionenes were shown to be the factors of effective control of PEC dissociation in water-salt solutions, with a diminished role of charge density upon lengthening the ionene chain. These polymer characteristics were also important for DNA-ionene PEC resistivity to DNase activity and the ability of ionenes to serve as gene delivery vectors in vitro and exhibited good correlation with the results of salt-induced dissociation of PEC. These data may be useful for developing correlations and mathematical models to predict synthetic gene delivery vector efficiency.     

Chloroplast biogenesis 92: In situ screening for divinyl chlorophyll(ide) a reductase mutants by spectrofluorometry

Chlorophyll biosynthetic heterogeneity is rooted mainly in parallel divinyl (DV) and monovinyl (MV) biosynthetic routes interconnected by 4-vinyl reductases (4VRs) that convert DV tetrapyrroles to MV tetrapyrroles by conversion of the vinyl group at position 4 of the macrocycle to ethyl. What is not clear at this stage is whether the various 4VR activities are catalyzed by one enzyme of broad specificity or by a family of enzymes encoded by one gene or multiple genes with each enzyme having narrow specificity. Additional research is needed to identify the various regulatory components of 4-vinyl reduction. In this undertaking, Arabidopsis mutants that accumulate DV chlorophyllide a and/or DV chlorophyll [Chl(ide)] a are likely to provide an appropriate resource. Because the Arabidopsis genome has been completely sequenced, the best strategy for identifying 4VR and/or putative regulatory 4VR genes is to screen Arabidopsis Chl mutants for DV Chl(ide) a accumulation. In wild-type Arabidopsis, a DV plant species, only MV chlorophyllide (Chlide) a is detectable. However in Chl mutants lacking 4VR activity, DV Chl(ide) a may accumulate in addition to MV Chl(ide) a. In the current work, an in situ assay of DV Chl(ide) a accumulation, suitable for screening a large number of mutants lacking 4-vinyl Chlide a reductase activity with minimal experimental handling, is described. The assay involves homogenization of the tissues in Tris-HCl:glycerol buffer and the recording of Soret excitation spectra at 77K. DV Chlide a formation is detected by a Soret excitation shoulder at 459 nm over a wide range of DV Chlide a/MV Chl a ratios. The DV Chlide a shoulder became undetectable at DV Chlide a/MV Chl a ratios less than 0.049, that is, at a DV Chlide a content of less than 5%.     

Muscle RING-finger protein-1 (MuRF1) as a connector of muscle energy metabolism and protein synthesis

During pathophysiological muscle wasting, a family of ubiquitin ligases, including muscle RING-finger protein-1 (MuRF1), has been proposed to trigger muscle protein degradation via ubiquitination. Here, we characterized skeletal muscles from wild-type (WT) and MuRF1 knockout (KO) mice under amino acid (AA) deprivation as a model for physiological protein degradation, where skeletal muscles altruistically waste themselves to provide AAs to other organs. When WT and MuRF1 KO mice were fed a diet lacking AA, MuRF1 KO mice were less susceptible to muscle wasting, for both myocardium and skeletal muscles. Under AA depletion, WT mice had reduced muscle protein synthesis, while MuRF1 KO mice maintained nonphysiologically elevated levels of skeletal muscle protein de novo synthesis. Consistent with a role of MuRF1 for muscle protein turnover during starvation, the concentrations of essential AAs, especially branched-chain AAs, in the blood plasma significantly decreased in MuRF1 KO mice under AA deprivation. To clarify the molecular roles of MuRF1 for muscle metabolism during wasting, we searched for MuRF1-associated proteins using pull-down assays and mass spectrometry. Muscle-type creatine kinase (M-CK), an essential enzyme for energy metabolism, was identified among the interacting proteins. Coexpression studies revealed that M-CK interacts with the central regions of MuRF1 including its B-box domain and that MuRF1 ubiquitinates M-CK, which triggers the degradation of M-CK via proteasomes. Consistent with MuRF1's role of adjusting CK activities in skeletal muscles by regulating its turnover in vivo, we found that CK levels were significantly higher in the MuRF1 KO mice than in WT mice. Glucocorticoid modulatory element binding protein-1 and 3-hydroxyisobutyrate dehydrogenase, previously identified as potential MuRF1-interacting proteins, were also ubiquitinated MuRF1-dependently. Taken together, these data suggest that, in a multifaceted manner, MuRF1 participates in the regulation of AA metabolism, including the control of free AAs and their supply to other organs under catabolic conditions, and in the regulation of ATP synthesis under metabolic-stress conditions where MuRF1 expression is induced.     

Adaptable TCR avidity thresholds for negative selection

Central tolerance plays a significant role in preventing autoimmune diseases by eliminating T cells with high and intermediate avidity for self. To determine the manner of setting the threshold for deletion, we created a unique transgenic mouse strain with a diverse T cell population and globally increased TCR avidity for self-peptide/MHC complexes. Despite the adaptations aimed at reducing T cell reactivity (reduced TCR levels and increased levels of TCR signaling inhibitor CD5), transgenic mice displayed more severe experimental allergic encephalomyelitis and lupus. The numbers and activity of natural (CD4(+)CD25(+)) regulatory T cells were not altered. These findings demonstrate that the threshold for deletion is adaptable, allowing survival of T cells with higher avidity when TCR avidity is globally increased.     

Anticipatory synergy adjustments in preparation to self-triggered perturbations in elderly individuals

We tested the ability of healthy elderly persons to use anticipatory synergy adjustments (ASAs) prior to a self-triggered perturbation of one of the fingers during a multifinger force production task. An index of a force-stabilizing synergy was computed reflecting covariation of commands to fingers. The subjects produced constant force by pressing with the four fingers of the dominant hand on force sensors against constant upwardly directed forces. The middle finger could be unloaded either by the subject pressing the trigger or unexpectedly by the experimenter. In the former condition, the synergy index showed a drop (interpreted as ASA) prior to the time of unloading. This drop started later and was smaller in magnitude as compared with ASAs reported in an earlier study of younger subjects. At the new steady state, a new sharing pattern of the force was reached. We conclude that aging is associated with a preserved ability to explore the flexibility of the mechanically redundant multifinger system but a decreased ability to use feedforward adjustments to self-triggered perturbations. These changes may contribute to the documented drop in manual dexterity with age.     

Physicochemical sequence characteristics that influence S-palmitoylation propensity

Over the past 30 years, several hundred eukaryotic proteins spanning from yeast to man have been shown to be S-palmitoylated. This post-translational modification involves the reversible addition of a 16-carbon saturated fatty acyl chain onto the cysteine residue of a protein where it regulates protein membrane association and distribution, conformation, and stability. However, the large-scale proteome-wide discovery of new palmitoylated proteins has been hindered by the difficulty of identifying a palmitoylation consensus sequence. Using a bioinformatics approach, we show that the enrichment of hydrophobic and basic residues, the cellular context of the protein, and the structural features of the residues surrounding the palmitoylated cysteine all influence the likelihood of palmitoylation. We developed a new palmitoylation predictor that incorporates these identified features, and this predictor achieves a Matthews Correlation Coefficient of .74 using 10-fold cross validation, and significantly outperforms existing predictors on unbiased testing sets. This demonstrates that palmitoylation sites can be predicted with accuracy by taking into account not only physiochemical properties of the modified cysteine and its surrounding residues, but also structural parameters and the subcellular localization of the modified cysteine. This will allow for improved predictions of palmitoylated residues in uncharacterized proteins. A web-based version of this predictor is currently under development.