Ancestry of the AUTS2 family–A novel group of polycomb-complex proteins involved in human neurological disease

Autism susceptibility candidate 2 (AUTS2) is a neurodevelopmental regulator associated with an autosomal dominant intellectual disability syndrome, AUTS2 syndrome, and is implicated as an important gene in human-specific evolution. AUTS2 exists as part of a tripartite gene family, the AUTS2 family, which includes two relatively undefined proteins, Fibrosin (FBRS) and Fibrosin-like protein 1 (FBRSL1). Evolutionary ancestors of AUTS2 have not been formally identified outside of the Animalia clade. A Drosophila melanogaster protein, Tay bridge, with a role in neurodevelopment, has been shown to display limited similarity to the C-terminal of AUTS2, suggesting that evolutionary ancestors of the AUTS2 family may exist within other Protostome lineages. Here we present an evolutionary analysis of the AUTS2 family, which highlights ancestral homologs of AUTS2 in multiple Protostome species, implicates AUTS2 as the closest human relative to the progenitor of the AUTS2 family, and demonstrates that Tay bridge is a divergent ortholog of the ancestral AUTS2 progenitor gene. We also define regions of high relative sequence identity, with potential functional significance, shared by the extended AUTS2 protein family. Using structural predictions coupled with sequence conservation and human variant data from 15,708 individuals, a putative domain structure for AUTS2 was produced that can be used to aid interpretation of the consequences of nucleotide variation on protein structure and function in human disease. To assess the role of AUTS2 in human-specific evolution, we recalculated allele frequencies at previously identified human derived sites using large population genome data, and show a high prevalence of ancestral alleles, suggesting that AUTS2 may not be a rapidly evolving gene, as previously thought.

Fractals in Action: An fMRI study on the Generation of new Hierarchical Levels in Motor Sequences

Generation of hierarchical structures, such as the embedding of subordinate elements into larger structures, is a core feature of human cognition. Discrimination of well-formed hierarchies is thought to rely on lateral prefrontal cortex (PFC). However, the brain bases underlying the active generation of new hierarchical levels remain poorly understood. Here, we created a new motor paradigm to isolate this active generative process. In fMRI, participants planned and performed (identical) movement sequences based on three previously learned rules: (1) a hierarchical ‘fractal’ rule that involved generation of new levels, (2) a linear ‘iterative’ rule adding items to existing hierarchical levels, and (3) simple ‘repetition’. We found that generation of new hierarchical levels (using the fractal rule) activated a bilateral motor planning-and imagery network, but did not involve lateral PFC. Conversely, adding items to existing hierarchical levels required M1 directly during execution. These results show that the generation of new hierarchical levels can be achieved without involvement of putative domain-general systems such as those ascribed to lateral PFC. We hypothesize that these systems might be important to parse hierarchical sequences in a multi-domain fashion but not necessarily to generate new hierarchical levels.

The crystal structure of PD1, aHaemophilussurface fibril domain

TheHaemophilussurface fibril (Hsf) is an unusually large trimeric autotransporter adhesin (TAA) expressed by the most virulent strains ofH. influenzae. Hsf is known to mediate adhesion between pathogen and host, allowing the establishment of potentially deadly diseases such as epiglottitis, meningitis and pneumonia. While recent research has suggested that this TAA might adopt a novel `hairpin-like' architecture, the characterization of Hsf has been limited toin silicomodelling and electron micrographs, with no high-resolution structural data available. Here, the crystal structure of Hsf putative domain 1 (PD1) is reported at 3.3 Å resolution. The structure corrects the previous domain annotation by revealing the presence of an unexpected N-terminal TrpRing domain. PD1 represents the first Hsf domain to be solved, and thus paves the way for further research on the `hairpin-like' hypothesis.

Structure of an atypical FeoB G-domain reveals a putative domain-swapped dimer

FeoB is a transmembrane protein involved in ferrous iron uptake in prokaryotic organisms. FeoB comprises a cytoplasmic soluble domain termed NFeoB and a C-terminal polytopic transmembrane domain. Recent structures of NFeoB have revealed two structural subdomains: a canonical GTPase domain and a five-helix helical domain. The GTPase domain hydrolyses GTP to GDP through a well characterized mechanism, a process which is required for Fe2+transport. In contrast, the precise role of the helical domain has not yet been fully determined. Here, the structure of the cytoplasmic domain of FeoB fromGallionella capsiferriformansis reported. Unlike recent structures of NFeoB, theG. capsiferriformansNFeoB structure is highly unusual in that it does not contain a helical domain. The crystal structures of both apo and GDP-bound protein forms a domain-swapped dimer.

Abstract 5289: Abnormally Tight Domain-Domain Interaction at Mutation Site Could be a Primary Cause of Catecholaminergic Polymorphic Ventricular Tachycardia: Insight from RyR2 S2246L/+ Knock-In Mouse Model

Mutations in cardiac ryanodine receptor 2 (RyR2) have been shown to be associated with catecholaminergic polymorphic ventricular tachycardia (CPVT). To study the underlying mechanism of this lethal arrhythmia, we developed knock-in (KI:RyR2 S2246L/+ ) mice model with the human CPVT-associated RyR2 mutation (S2246L). The KI mice revealed no structural or histological abnormality in hearts, and also had no contractile or relaxation dysfunction at rest. In all KI mice (n=6), however, bi-directional ventricular tachycardia (VT) was observed after exercise with treadmill (Ex:6/6), but not observed in wild-type (WT) mice (Ex:0/7). In isolated cardiomyocytes, line scan images were obtained to measure local Ca 2+ release events using a confocal microscopy with fluo-4 AM as a Ca 2+ indicator. In the KI cardiomyocytes, the frequency of Ca 2+ sparks (SpF: s −1 ·100μm −1 ) was much more increased in response to 100 nM isoproterenol than in WT cardiomyocytes (KI:4.7±0.5 vs WT:1.9±0.2, p<0.01). Using the canine cardiac SR, we fluorescently labeled RyR2 with methylcoumarin acetamido (MCA), using either DP 2232–2266 or DP 2232–2266 mut; Ser is mutated to Leu (S2246L), as a carrier; DP 2232–2266 harbors the same CPVT mutation site as KI mice (S2246L). The binding affinity of DP 2232–2266 mut to RyR2 (Kd=0.08μM) was higher than that of DP 2232–2266 (Kd=0.32μM), suggesting that abnormally tight interaction of the domain pair (between the domain2232–2266 and another putative domain) may be formed by the S2246L mutation. Interestingly, addition of DP 2232–2266 to the SR dose-dependently inhibited the cAMP (30μM)-induced Ca 2+ leak; IC50=0.1μM, although DP 2232–2266 had no effect on the cAMP-induced increase in 2808Ser phosphorylation. In conclusion, some type of RyR2 mutation in CPVT may causatively induce hyper-activated channel gating by forming abnormally tight domain-domain interaction, triggering diastolic Ca 2+ release and hence lethal arrhythmia. Interruption of such abnormal domain-domain interaction (by competing with native domain) may lead to a new therapeutic strategy against CPVT.

Functional Organization of the Autotransporter Adhesin Involved in Diffuse Adherence

ABSTRACT The Escherichia coli adhesin involved in diffuse adherence (AIDA-I) is a multifunctional autotransporter protein that mediates bacterial aggregation and biofilm formation, as well as adhesion and invasion of cultured epithelial cells. To elucidate the structure-function relationships of AIDA-I, we performed transposon-based linker scanning mutagenesis and constructed mutants with site-directed deletions. Twenty-nine different mutants with insertions that did not affect protein expression were obtained. Eleven mutants were deficient for one or two but not all of the functions associated with the expression of AIDA-I. Functional characterization of the transposon mutants and of an additional deletion mutant suggested that the N-terminal third of mature AIDA-I is involved in binding of this protein to cultured epithelial cells. The purified product of the putative domain could bind to cultured epithelial cells, confirming the importance of this region in adhesion. We also identified several different mutants in which invasion and adhesion were changed to different extents and two mutants in which autoaggregation and biofilm formation were also affected differently. These results suggest that although conceptually linked, adhesion and invasion, as well as autoaggregation and biofilm formation, are phenomena that may rely on distinct mechanisms when they are mediated by AIDA-I. This study sheds new light on the workings of a protein belonging to an emerging family of strikingly versatile virulence factors.

The dynamics of chromatin condensation: redistribution of topoisomerase II in the 87A7 heat shock locus during induction and recovery.

We have examined the in vivo sites of action for topoisomerases II in the 87A7 heat shock locus as a function of gene activity. When the hsp70 genes are induced, there is a dramatic redistribution of topoisomerase II in the locus which parallels many of the observed alterations in chromatin structure. In addition to changes in the topoisomerase II distribution within the locus, we find topoisomerase II localized around the putative domain boundaries scs and scs'. During recovery, when the chromatin fiber of the locus recondenses, the major sites of action for topoisomerase II appear to be located within the two hsp70 genes and in the intergenic spacer separating the two genes.

The dynamics of chromatin condensation: redistribution of topoisomerase II in the 87A7 heat shock locus during induction and recovery

We have examined the in vivo sites of action for topoisomerases II in the 87A7 heat shock locus as a function of gene activity. When the hsp70 genes are induced, there is a dramatic redistribution of topoisomerase II in the locus which parallels many of the observed alterations in chromatin structure. In addition to changes in the topoisomerase II distribution within the locus, we find topoisomerase II localized around the putative domain boundaries scs and scs'. During recovery, when the chromatin fiber of the locus recondenses, the major sites of action for topoisomerase II appear to be located within the two hsp70 genes and in the intergenic spacer separating the two genes.