One third of inherited genetic diseases are caused by mRNAs harboring premature termination codons as a result of nonsense mutations. These aberrant mRNAs are degraded by the Nonsense-Mediated mRNA Decay (NMD) pathway. A central component of the NMD pathway is Upf1, an RNA-dependent ATPase and helicase. Upf1 is a known phosphorylated protein, but only portions of this large protein have been examined for phosphorylation sites and the functional relevance of its phosphorylation has not been elucidated in Saccharomyces cerevisiae. Using tandem mass spectrometry analyses, we report the identification of 11 putative phosphorylated sites in S. cerevisiae Upf1. Five of these phosphorylated residues are located within the ATPase and helicase domains and are conserved in higher eukaryotes, suggesting a biological significance for their phosphorylation. Indeed, functional analysis demonstrated that a small carboxy-terminal motif harboring at least three phosphorylated amino acids is important for three Upf1 functions: ATPase activity, NMD activity and the ability to promote translation termination efficiency. We provide evidence that two tyrosines within this phospho-motif (Y-738 and Y-742) act redundantly to promote ATP hydrolysis, NMD efficiency and translation termination fidelity.
Many pathogens, such as Pseudomonas aeruginosa and Escherichia coli bacteria can easily attach to surfaces and form stable biofilms. The formation of such biofilms in surfaces presents a problem in environmental, biomedical, and industrial processes, among many others. Aiming to provide a plausible solution to this issue, the anionic and hydrophobic peptide Maximin H5 C-terminally deaminated isoform (MH5C) has been modified with a cysteine in the C-terminal (MH5C-Cys) and coupled to polyethylene glycol (PEG) polymers of varying sizes (i.e., 2 kDa and 5 kDa) to serve as a surface protective coating. Briefly, the MH5C-Cys was bioconjugated to PEG and purified by size exclusion chromatography while the reaction was confirmed via SDS-PAGE and MALDI ToF. Moreover, the preventive antimicrobial activity of the MH5C-Cys-PEG conjugates was performed via the growth curves method, showing inhibition of bacterial growth after 24 h. The efficacy of these peptide−polymer conjugates was extensively characterized via scanning electron microscopy (SEM), minimum inhibition concentration (MIC), minimum biofilm inhibition concentration (MBIC), and minimum biofilm eradication concentration (MBEC) assays to evaluate their ability to eradicate and prevent the biofilms. Interestingly, this work demonstrated a critical PEG polymer weight of 5 kDa as ideal when coupled to the peptide to achieve inhibition and eradication of the biofilm formation in both bacteria strains. According to the MICs (40 μM) and MBICs (300 μM), we can conclude that this conjugate (MH5C-Cys-5 kDa) has an action that prevents/inhibits the formation of biofilms and the eradication of biofilms (MBEC 500 μM). In contrast, the MH5C-Cys peptide with PEG polymer of 2 kDa did not show inhibition or eradication of the biofilms.
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