Lysine acetylome profiling uncovers novel histone deacetylase substrate proteins in Arabidopsis

Histone deacetylases have central functions in regulating stress defenses and development in plants. However, the knowledge about the deacetylase functions is largely limited to histones, although these enzymes were found in diverse subcellular compartments. In this study, we determined the proteome‐wide signatures of the RPD3/HDA1 class of histone deacetylases in Arabidopsis. Relative quantification of the changes in the lysine acetylation levels was determined on a proteome‐wide scale after treatment of Arabidopsis leaves with deacetylase inhibitors apicidin and trichostatin A. We identified 91 new acetylated candidate proteins other than histones, which are potential substrates of the RPD3/HDA1‐like histone deacetylases in Arabidopsis, of which at least 30 of these proteins function in nucleic acid binding. Furthermore, our analysis revealed that histone deacetylase 14 (HDA14) is the first organellar‐localized RPD3/HDA1 class protein found to reside in the chloroplasts and that the majority of its protein targets have functions in photosynthesis. Finally, the analysis of HDA14 loss‐of‐function mutants revealed that the activation state of RuBisCO is controlled by lysine acetylation of RuBisCO activase under low‐light conditions.     

Cytochemical and molecular characteristics of the process of cornification during feather morphogenesis

Feathers are the most complex epidermal derivatives among vertebrates. The present review deals with the origin of feathers from archosaurian reptiles, the cellular and molecular aspects of feather morphogenesis, and focus on the synthesis of keratins and associated proteins. Feathers consist of different proteins among which exists a specialized group of small proteins called beta-keratins. Genes encoding these proteins in the chick genome are distributed in different chromosomes, and most genes encode for feather keratins. The latter are here recognized as proteins associated with the keratins of intermediate filaments, and functionally correspond to keratin-associated proteins of hairs, nails and horns in mammals. These small proteins possess unique properties, including resistance and scarce elasticity, and were inherited and modified in feathers from ancestral proteins present in the scales of archosaurian progenitors of birds. The proteins share a common structural motif, the core box, which was present in the proteins of the reptilian ancestors of birds. The core box allows the formation of filaments with a different molecular mechanism of polymerization from that of alpha-keratins. Feathers evolved after the establishment of a special morphogenetic mechanism gave rise to barb ridges. During development, the epidermal layers of feathers fold to produce barb ridges that produce the ramified structure of feathers. Among barb ridge cells, those of barb and barbules initially accumulate small amounts of alpha-keratins that are rapidly replaced by a small protein indicated as “feather keratin”. This 10 kDa protein becomes the predominant form of corneous material of feathers. The main characteristics of feather keratins, their gene organization and biosynthesis are similar to those of their reptilian ancestors. Feather keratins allow elongation of feather cells among supportive cells that later degenerate and leave the ramified microstructure of barbs. In downfeathers, barbs are initially independent and form plumulaceous feathers that rest inside a follicle. Stem cells remain in the follicle and are responsible for the regeneration of pennaceous feathers. New barb ridges are produced and they merge to produce a rachis and a flat vane. The modulation of the growth pattern of barb ridges and their fusion into a rachis give rise to a broad variety of feather types, including asymmetric feathers for flight. Feather morphogenesis suggests possible stages for feather evolution and diversification from hair-like outgrowths of the skin found in fossils of pro-avian archosaurians.     

The importance of being kinked: role of Pro residues in the selectivity of the helical antimicrobial peptide P5

Antimicrobial peptides (AMPs) are promising compounds for developing new antibiotic drugs against drug‐resistant bacteria. Many of them kill bacteria by perturbing their membranes but exhibit no significant toxicity towards eukaryotic cells. The identification of the features responsible for this selectivity is essential for their pharmacological development. AMPs exhibit few conserved features, but a statistical analysis of an AMP sequence database indicated that many α‐helical AMPs surprisingly have a helix‐breaking Pro residue in the middle of their sequence. To discriminate among the different possible hypotheses for the functional role of this feature, we designed an analogue of the antimicrobial peptide P5, in which the central Pro was deleted (analogue P5Del). Pro removal resulted in a dramatic increase of toxicity. This was explained by the observation that P5Del binds both charged and neutral membranes, whereas P5 has no appreciable affinity towards neutral bilayers. CD and simulative data provided a rationalization of this behavior. In solution P5, due to the presence of Pro, attains compact conformations, in which its apolar residues are partially shielded from the solvent, whereas P5Del is more helical. These structural differences reduce the hydrophobic driving force for association of P5 to neutral membranes, whereas its binding to anionic bilayers can still take place because of electrostatic attraction. After membrane binding, the Pro residue does not preclude the attainment of a membrane‐active amphiphilic helical conformation. These findings shed light on the role of Pro residues in the selectivity of AMPs and provide hints for the design of new, highly selective compounds. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Metabolic analysis of the synthesis of high levels of intracellular human SOD in Saccharomyces cerevisiae rhSOD 2060 411 SGA122

The synthesis of human superoxide dismutase (SOD) in batch cultures of a Saccharomyces cerevisiae strain using a glucose-limited minimal medium was studied through metabolic flux analysis. A stoichiometric model was built, which included 78 reactions, according to metabolic pathways operative in these strains during respirofermentative and oxidative metabolism. It allowed calculation of the distribution of metabolic fluxes during diauxic growth on glucose and ethanol. Fermentation profiles and metabolic fluxes were analyzed at different phases of diauxic growth for the recombinant strain (P+) and for its wild type (P-). The synthesis of SOD by the strain P+ resulted in a decrease in specific growth rate of 34 and 54% (growth on glucose and ethanol respectively) in comparison to the wild type. Both strains exhibited similar flux of glucose consumption and ethanol synthesis but important differences in carbon distribution with biomass/substrate yields and ATP production 50% higher in P-. A higher contribution of fermentative metabolism, with 64% of the energy produced at the phosphorylation level, was observed during SOD production. The flux of precursors to amino acids and nucleotides was higher in the recombinant strain, in agreement with the higher total RNA and protein levels. Lower specific growth rates in strain P+ appear to be related to the decrease in the rate of synthesis of nonrecombinant protein, as well as a decrease in the activities of the pentose phosphate (PP) pathway and TCA cycle. A very different way of entry into the stationary phase was observed for each strain: in the wild-type strain most metabolic fluxes decreased and fluxes related to energy reserve synthesis increased, while in the P+ strain the flux of 22 reactions (including PP pathway and amino acids biosynthesis) related to SOD production increased their fluxes. Changes in SOD production rates at different physiological states appear to be related to the differences in building blocks availability between respirofermentative and oxidative metabolism. Using the present expression system, ideal conditions for SOD synthesis are represented by either active growth during respirofermentative metabolism or transition from a growing to a nongrowing state. An increase in SOD flux could be achieved using an expression system nonassociated to growth and potentially eliminating part of the metabolic burden.     

M234Glu is a component of the proton sponge in the reaction center from photosynthetic bacteria

Bacterial reaction centers use light energy to couple the uptake of protons to the successive semi-reduction of two quinones, namely Q_A and Q_B. These molecules are situated symmetrically in regard to a non-heme iron atom. Four histidines and one glutamic acid, M234Glu, constitute the five ligands of this atom. By flash-induced absorption spectroscopy and delayed fluorescence we have studied in the M234EH and M234EL variants the role played by this acidic residue on the energetic balance between the two quinones as well as in proton uptake. Delayed fluorescence from the P⁺Q_A^? state (P is the primary electron donor) and temperature dependence of the rate of P⁺Q_A^? charge recombination that are in good agreement show that in the two RC variants, both Q_A^? and Q_B^? are destabilized by about the same free energy amount: respectively ～ 100 ± 5 meV and 90 ± 5 meV for the M234EH and M234EL variants, as compared to the WT. Importantly, in the M234EH and M234EL variants we observe a collapse of the high pH band (present in the wild-type reaction center) of the proton uptake amplitudes associated with formation of Q_A^? and Q_B^?. This band has recently been shown to be a signature of a collective behaviour of an extended, multi-entry, proton uptake network. M234Glu seems to play a central role in the proton sponge-like system formed by the RC protein.     

Root Hairs as a Model System for Studying Plant Cell Growth

Root hairs are tip-growing projections that form on specializedepidermal cells. Physiological studies are identifying key transportersrequired for hair growth, and drug studies have been instructivein defining the role of the cytoskeleton in cell morphogenesis.Genetic analysis is identifying proteins involved in cell growthand the phenotypes of the mutants are instructive in definingthe precise function of these proteins in cellular morphogenesis.Recent progress in our understandings of cell growth using thearabidopsis root hair as a model system is reviewed. Copyright2001 Annals of Botany Company Arabidopsis, root hair, trichoblast, actin, microtubules, cell wall, genetics, calcium, potassium, phosphorus     

Impact of antigen presentation on TCR modulation and cytokine release: implications for detection and sorting of antigen-specific CD8+ T cells using HLA-A2 wild-type or HLA-A2 mutant tetrameric complexes

Soluble MHC class I molecules loaded with antigenic peptides are available either to detect and to enumerate or, alternatively, to sort and expand MHC class I-restricted and peptide-reactive T cells. A defined number of MHC class I/peptide complexes can now be implemented to measure T cell responses induced upon Ag-specific stimulation, including CD3/CD8/zeta-chain down-regulation, pattern, and quantity of cytokine secretion. As a paradigm, we analyzed the reactivity of a Melan-A/MART-1-specific and HLA-A2-restricted CD8(+) T cell clone to either soluble or solid-phase presented peptides, including the naturally processed and presented Melan-A/MART-1 peptide AAGIGILTV or the peptide analog ELAGIGILTV presented either by the HLA-A2 wild-type (wt) or mutant (alanineright arrowvaline aa 245) MHC class I molecule, which reduces engagement of the CD8 molecule with the HLA-A2 heavy chain. Soluble MHC class I complexes were used as either monomeric or tetrameric complexes. Soluble monomeric MHC class I complexes, loaded with the Melan-A/MART-1 peptide, resulted in CD3/CD8 and TCR zeta-chain down-regulation, but did not induce measurable cytokine release. In general, differences pertaining to CD3/CD8/zeta-chain regulation and cytokine release, including IL-2, IFN-gamma, and GM-CSF, were associated with 1) the format of Ag presentation (monomeric vs tetrameric MHC class I complexes), 2) wt vs mutant HLA-A2 molecules, and 3) the target Ag (wt vs analog peptide). These differences are to be considered if T cells are exposed to recombinant MHC class I Ags loaded with peptides implemented for detection, activation, or sorting of Ag-specific T cells.     

Characterizing the complexity of enzymes on the basis of their mechanisms and structures with a bio-computational analysis

Enzymes are basically composed of 20 naturally occurring amino acids, yet they catalyse a dizzying array of chemical reactions, with regiospecificity and stereospecificity and under physiological conditions. In this review, we attempt to gain some understanding of these complex proteins, from the chemical versatility of the catalytic toolkit, including the use of cofactors (both metal ions and organic molecules), to the complex mapping of reactions to proteins (which is rarely one-to-one), and finally the structural complexity of enzymes and their active sites, often involving multidomain or multisubunit assemblies. This work highlights how the enzymes that we see today reflect millions of years of evolution, involving de novo design followed by exquisite regulation and modulation to create optimal fitness for life.