A combinatorial native MS and LC-MS/MS approach reveals high intrinsic phosphorylation of human Tau but minimal levels of other key modifications

Abnormal changes of neuronal Tau protein, such as phosphorylation and aggregation, are considered hallmarks of cognitive deficits in Alzheimer''s disease. Abnormal phosphorylation is thought to precede aggregation and therefore to promote aggregation, but the nature and extent of phosphorylation remain ill-defined. Tau contains ∼85 potential phosphorylation sites, which can be phosphorylated by various kinases because the unfolded structure of Tau makes them accessible. However, methodological limitations (e.g. in MS of phosphopeptides, or antibodies against phosphoepitopes) led to conflicting results regarding the extent of Tau phosphorylation in cells. Here we present results from a new approach based on native MS of intact Tau expressed in eukaryotic cells (Sf9). The extent of phosphorylation is heterogeneous, up to ∼20 phosphates per molecule distributed over 51 sites. The medium phosphorylated fraction P_m showed overall occupancies of ∼8 P_i (± 5) with a bell-shaped distribution; the highly phosphorylated fraction P_h had 14 P_i (± 6). The distribution of sites was highly asymmetric (with 71% of all P-sites in the C-terminal half of Tau). All sites were on Ser or Thr residues, but none were on Tyr. Other known posttranslational modifications were near or below our detection limit (e.g. acetylation, ubiquitination). These findings suggest that normal cellular Tau shows a remarkably high extent of phosphorylation, whereas other modifications are nearly absent. This implies that abnormal phosphorylations at certain sites may not affect the extent of phosphorylation significantly and do not represent hyperphosphorylation. By implication, the pathological aggregation of Tau is not likely a consequence of high phosphorylation.     

Genetically encoded impairment of neuronal KCC2 cotransporter function in human idiopathic generalized epilepsy

The KCC2 cotransporter establishes the low neuronal Cl⁻ levels required for GABA_A and glycine (Gly) receptor-mediated inhibition, and KCC2 deficiency in model organisms results in network hyperexcitability. However, no mutations in KCC2 have been documented in human disease. Here, we report two non-synonymous functional variants in human KCC2, R952H and R1049C, exhibiting clear statistical association with idiopathic generalized epilepsy (IGE). These variants reside in conserved residues in the KCC2 cytoplasmic C-terminus, exhibit significantly impaired Cl⁻-extrusion capacities resulting in less hyperpolarized Gly equilibrium potentials (E_Gly), and impair KCC2 stimulatory phosphorylation at serine 940, a key regulatory site. These data describe a novel KCC2 variant significantly associated with a human disease and suggest genetically encoded impairment of KCC2 functional regulation may be a risk factor for the development of human IGE.     

Arabidopsis KINETOCHORE NULL2 Is an Upstream Component for Centromeric Histone H3 Variant cenH3 Deposition at Centromeres

The centromeric histone H3 variant cenH3 is an essential centromeric protein required for assembly, maintenance, and proper function of kinetochores during mitosis and meiosis. We identified a KINETOCHORE NULL2 (KNL2) homolog in Arabidopsis thaliana and uncovered features of its role in cenH3 loading at centromeres. We show that Arabidopsis KNL2 colocalizes with cenH3 and is associated with centromeres during all stages of the mitotic cell cycle, except from metaphase to mid-anaphase. KNL2 is regulated by the proteasome degradation pathway. The KNL2 promoter is mainly active in meristematic tissues, similar to the cenH3 promoter. A knockout mutant for KNL2 shows a reduced level of cenH3 expression and reduced amount of cenH3 protein at chromocenters of meristematic nuclei, anaphase bridges during mitosis, micronuclei in pollen tetrads, and 30% seed abortion. Moreover, knl2 mutant plants display reduced expression of suppressor of variegation 3-9 homologs2, 4, and 9 and reduced DNA methylation, suggesting an impact of KNL2 on the epigenetic environment for centromere maintenance.     

The Smallest Known Genomes of Multicellular and Toxic Cyanobacteria: Comparison,Minimal Gene Sets for Linked Traits and the Evolutionary Implications

Cyanobacterial morphology is diverse, ranging from unicellular spheres or rods to multicellular structures such as colonies and filaments. Multicellular species represent an evolutionary strategy to differentiate and compartmentalize certain metabolic functions for reproduction and nitrogen (N₂) fixation into specialized cell types (e.g. akinetes, heterocysts and diazocytes). Only a few filamentous, differentiated cyanobacterial species, with genome sizes over 5 Mb, have been sequenced. We sequenced the genomes of two strains of closely related filamentous cyanobacterial species to yield further insights into the molecular basis of the traits of N₂ fixation, filament formation and cell differentiation. Cylindrospermopsis raciborskii CS-505 is a cylindrospermopsin-producing strain from Australia, whereas Raphidiopsis brookii D9 from Brazil synthesizes neurotoxins associated with paralytic shellfish poisoning (PSP). Despite their different morphology, toxin composition and disjunct geographical distribution, these strains form a monophyletic group. With genome sizes of approximately 3.9 (CS-505) and 3.2 (D9) Mb, these are the smallest genomes described for free-living filamentous cyanobacteria. We observed remarkable gene order conservation (synteny) between these genomes despite the difference in repetitive element content, which accounts for most of the genome size difference between them. We show here that the strains share a specific set of 2539 genes with >90% average nucleotide identity. The fact that the CS-505 and D9 genomes are small and streamlined compared to those of other filamentous cyanobacterial species and the lack of the ability for heterocyst formation in strain D9 allowed us to define a core set of genes responsible for each trait in filamentous species. We presume that in strain D9 the ability to form proper heterocysts was secondarily lost together with N₂ fixation capacity. Further comparisons to all available cyanobacterial genomes covering almost the entire evolutionary branch revealed a common minimal gene set for each of these cyanobacterial traits.     

Ectopic myelinating oligodendrocytes in the dorsal spinal cord as a consequence of altered semaphorin 6D signaling inhibit synapse formation

Different types of sensory neurons in the dorsal root ganglia project axons to the spinal cord to convey peripheral information to the central nervous system. Whereas most proprioceptive axons enter the spinal cord medially, cutaneous axons typically do so laterally. Because heavily myelinated proprioceptive axons project to the ventral spinal cord, proprioceptive axons and their associated oligodendrocytes avoid the superficial dorsal horn. However, it remains unclear whether their exclusion from the superficial dorsal horn is an important aspect of neural circuitry. Here we show that a mouse null mutation of Sema6d results in ectopic placement of the shafts of proprioceptive axons and their associated oligodendrocytes in the superficial dorsal horn, disrupting its synaptic organization. Anatomical and electrophysiological analyses show that proper axon positioning does not seem to be required for sensory afferent connectivity with motor neurons. Furthermore, ablation of oligodendrocytes from Sema6d mutants reveals that ectopic oligodendrocytes, but not proprioceptive axons, inhibit synapse formation in Sema6d mutants. Our findings provide new insights into the relationship between oligodendrocytes and synapse formation in vivo, which might be an important element in controlling the development of neural wiring in the central nervous system.     

Detection of heme oxygenase activity in a library of four-helix bundle proteins: towards the de novo synthesis of functional heme proteins

Design and chemical synthesis of de novo heme proteins with enzymatic activity on cellulose membranes is described. 352 antiparallel four-helix bundle proteins with a single histidine for heme ligation were assembled from three different sets of short amphipathic helices on membrane-bound peptide templates. The templates were coupled by linkers to cellulose membranes of microplate format, which could be cleaved for control of intermediate and final products. The incorporation of heme and the heme oxygenase activity of the 352 proteins were monitored by measuring UV-visible spectra directly on the cellulose. The kinetics of the heme oxygenase reaction was studied by monitoring the decrease of the Soret band and the transient absorbance of verdoheme being an intermediate product in the formation of biliverdin. Four of the proteins covering a broad range of the enzymatic rate constants were selected and synthesized in solution for further characterization. Detailed studies by redox potentiometry, analytical ultracentrifugation, and electron paramagnetic resonance spectroscopy yielded information about the aggregation state of the proteins, the spin state and the putative coordination environment of the iron. The amount of five-coordinated high-spin iron and a positive reduction potential were found to promote the oxygenase activity of the proteins.