Matrix metalloproteinase-9 (MMP-9) plays an important role in both physiological and pathological processes. This enzyme is a peripheral biomarker of neuroinflammation in multiple sclerosis (MS), a chronic autoimmune disease of the central nervous system. Presently, expensive magnetic resonance imaging (MRI) studies are used to monitor subclinical disease activity in MS. An alternative to costly MRI scans could be the detection of MMP-9, using a low-cost, disposable sensor system for MMP-9 suitable for home-monitoring of inflammation. This would allow an early prediction of the failure of anti-inflammatory therapies and more timely clinical intervention to limit neuronal damage and prevent disability. Herein we present the development of a disposable sensor for fast and straightforward detection of MMP-9. Biosensors were produced by coating electrodes with oxidized dextran and subsequent cross-linking with peptides containing specific cleavage sites for MMP-9. Exposure of the films to the enzyme resulted in the degradation of the films, which was monitored using impedance measurements. Sensor response was rapid, a significant impedance change was usually observed within 5 min after the addition of MMP-9. Sensors showed a negligible response to matrix metalloproteinase-2 (MMP-2), a protease which may interfere with MMP-9 detection. The peptide sequence with the highest sensitivity and selectivity Leu-Gly-Arg-Met-Gly-Leu-Pro-Gly-Lys was selected to construct calibration curves. MMP-9 was successfully detected in a clinically relevant range from 50 to 400 ng/ml. Two different processes of hydrogel degradation were observed on electrode surfaces with different roughness, and both appeared suitable to monitor MMP-9 activity. The sensor materials are generic and can be easily adopted to respond to other proteases by selecting peptide cross-linkers with suitable cleavage sites.

The highly conserved Elongator complex is a translational regulator that plays a critical role in neurodevelopment, neurological diseases, and brain tumors. Numerous clinically relevant variants have been reported in the catalytic Elp123 subcomplex, while no missense mutations in the accessory subcomplex Elp456 have been described. Here, we identify ELP4 and ELP6 variants in patients with developmental delay, epilepsy, intellectual disability, and motor dysfunction. We determine the structures of human and murine Elp456 subcomplexes and locate the mutated residues. We show that patient-derived mutations in Elp456 affect the tRNA modification activity of Elongator in vitro as well as in human and murine cells. Modeling the pathogenic variants in mice recapitulates the clinical features of the patients and reveals neuropathology that differs from the one caused by previously characterized Elp123 mutations. Our study demonstrates a direct correlation between Elp4 and Elp6 mutations, reduced Elongator activity, and neurological defects. Foremost, our data indicate previously unrecognized differences of the Elp123 and Elp456 subcomplexes for individual tRNA species, in different cell types and in different key steps during the neurodevelopment of higher organisms.

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Transfer RNA (tRNA) molecules are essential to decode messenger RNA codons during protein synthesis. All known tRNAs are heavily modified at multiple positions through post-transcriptional addition of chemical groups. Modifications in the tRNA anticodons are directly influencing ribosome decoding and dynamics during translation elongation and are crucial for maintaining proteome integrity. In eukaryotes, wobble uridines are modified by Elongator, a large and highly conserved macromolecular complex. Elongator consists of two subcomplexes, namely Elp123 containing the enzymatically active Elp3 subunit and the associated Elp456 hetero-hexamer. The structure of the fully assembled complex and the function of the Elp456 subcomplex have remained elusive. Here, we show the cryo-electron microscopy structure of yeast Elongator at an overall resolution of 4.3 Å. We validate the obtained structure by complementary mutational analyses in vitro and in vivo. In addition, we determined various structures of the murine Elongator complex, including the fully assembled mouse Elongator complex at 5.9 Å resolution. Our results confirm the structural conservation of Elongator and its intermediates among eukaryotes. Furthermore, we complement our analyses with the biochemical characterization of the assembled human Elongator. Our results provide the molecular basis for the assembly of Elongator and its tRNA modification activity in eukaryotes.

Elp3, the catalytic subunit of the eukaryotic Elongator complex, is a lysine acetyltransferase that acetylates the C5 position of wobble-base uridines (U34) in transfer RNAs (tRNAs). This Elongator-dependent RNA acetylation of anticodon bases affects the ribosomal translation elongation rates and directly links acetyl-CoA metabolism to both protein synthesis rates and the proteome integrity. Of note, several human diseases, including various cancers and neurodegenerative disorders, correlate with the dysregulation of Elongator’s tRNA modification activity. In this review, we focus on recent findings regarding the structure of Elp3 and the role of acetyl-CoA during its unique modification reaction.