Natural biological enzymes possess catalytic sites that are generally surrounded by a large three-dimensional scaffold. However, the proportion of the protein molecule that participates in the catalytic reaction is relatively small. The generation of artificial or miniature enzymes has long been a focus of research because enzyme mimetics can be produced with high activity at low cost. These enzymes aim to mimic the active sites without the additional architecture contributed by the protein chain. Previous work has shown that amyloidogenic peptides are able to self-assemble to create an active site that is capable of binding zinc and catalysing an esterase reaction. Here, we describe the structural characterisation of a set of designed peptides that form an amyloid-like architecture and reveal that their capability to mimic carbonic anhydrase and serve as enzyme-like catalysts is related to their ability to self-assemble. These amyloid fibril structures can bind the metal ion Znvia a three-dimensional arrangement of His residues created by the amyloid architecture. Our results suggest that the catalytic efficiency of amyloid-like assembly is not only zinc-dependent but also depends on an active centre created by the peptides which is, in turn, dependent on the ordered architecture. These fibrils have good esterase activity, and they may serve as good models for the evolution of modern-day enzymes. Furthermore, they may be useful in designing self-assembling fibrils for applications as metal ion catalysts. This study also demonstrates that the ligands surrounding the catalytic site affect the affinity of the zinc-binding site to bind the substrate contributing to the enzymatic activity of the assembled peptides.

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Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is caused by mutations in SACS, which manifest as a childhood-onset cerebellar ataxia. Cellular ARSACS phenotypes include mitochondrial dysfunction, intermediate filament (IF) disorganization, and loss of Purkinje neurons. It is unclear how the loss of SACS causes these deficits, or why they manifest as cerebellar ataxia. We employed a multi-omics approach to characterize molecular and cellular deficiencies in SACS knockout (KO) cells. We identified alterations in microtubule structure and dynamics, protein trafficking, and mislocalization of synaptic and focal adhesion proteins. Targeting PTEN, a negative regulator of focal adhesions, rescued several cellular phenotypes in SACS KO cells. We found sacsin interacts with proteins implicated in vesicle transport, including HSP proteins, and interactions between structural and cell adhesion proteins were diminished in SACS KO cells. In all, this study suggests that trafficking and localization of synaptic adhesion proteins is a causal molecular deficiency in ARSACS.

We employ targeted mass spectrometry to compare the levels of H2AX S139 phosphorylation (γH2AX) and Y142 phosphorylation. We use synthetic peptides to facilitate MS optimization and estimate relative detection efficiencies for the different modifications. Despite phosphopeptide enrichment from large amounts of starting material, we are unable to detect endogenous H2AX Y142 phosphorylation, indicating that it is present at low abundance (<1%). We also calculate the relative levels of H2AX compared to other H2A isoforms and quantify the proportion of H2AX that is phosphorylated on S139 (γH2AX) after ionizing radiation. Introduction S139 phosphorylationThe histone H2A family variant, H2AX, is distinguished from canonical H2A family members through a 22 amino acid C-terminal tail [1]. Phosphorylation of the C-terminal domain of H2AX at position 139 (γH2AX) is a rapid response to DNA double-strand breaks (DSB). S139 is phosphorylated by ATM, ATR and DNA-PK, which are phosphatidylinositol 3-kinase-related kinases. γH2AX foci are widely used as diagnostic markers of DSB. The utility of γH2AX as a marker stems from the rapid (<1 min) and extensive nature of this modification. Rogakou et al. observed that approximately 1% of total H2AX becomes phosphorylated per gray of ionizing radiation (IR), and extrapolated from H2AX relative abundance that each DSB results in γH2AX covering on average 2 million bp [2]. The biological function of such large γH2AX domains is not clear, and the H2AX histone is not essential for DSB repair, however H2AX -/-mice show increased ionizing radiation sensitivity, as well as increases in chromatid breaks and dicentric chromosomes [3]. Y142 phosphorylationThe H2AX C-terminal domain can also be phosphorylated on tyrosine 142 by the WSTF remodelling factor kinase [4][5][6]. Cook et al. show that dephosphorylation of Y142 upon DNA damage avoids apoptosis. Using synthetic phosphopeptides, they demonstrate binding of pro-apoptotic factors to S139 Y142 doubly-phosphorylated peptides: the implication is that Y142 phosphorylation is abundant, and will be located in proximity to DNA damage. While kinases and phosphatases responsible for creating and removing this modification have been identified, the basal level of Y142 phosphorylation is unknown, although our earlier intact histone MS analysis indicates that in HeLa cells it is not greater than~10% [7]. Scully et al. expressed epitope-tagged H2AX in H2AX -/-mouse ES cells and identified a number of H2AX modifications by mass spectrometry, including S139 and T101 phosphorylation, however Y142 phosphorylation was not detected [8]. The role of Y142ph in the DNA damage response is of great interest, with the identification of putative interacting proteins that recognise the doubly phosphorylated C-terminal tail [9]. Mutation of these residues has been carried out in the chicken DT40 cell line and revealed that Y142A IR sensitivity is rescued by co-mutation of S139A [10]. H2AX levels across cell lines and in the genomeGiven the role of H2AX phospho...