Radziwon, K. | Search bioRxiv

bioRxiv2025

Phosphoproteome-derived peptide libraries for deep specificity profiling of phosphatases and phospholyases

Protein phosphorylation is dynamically regulated by the opposing activities of phosphowriter enzymes (kinases) and phosphoeraser enzymes (phosphatases and phospholyases). While significant progress has been made toward defining the sequences preferences of kinases, the selectivity of phosphoerasers has not been explored at scale. Here, we develop an experimental platform based on tandem mass spectrometry analysis of phosphoproteome-derived peptide libraries (PhosPropels) to map phosphoeraser activity across thousands of biologically relevant phosphosites. We extract positional residue preferences to rapidly define sequence motifs recognized by eight phosphoerasers spanning diverse species of origin, protein folds, and enzymatic mechanisms. Taking advantage of the throughput of our approach, we profiled 34 variants of the phosphothreonine lyase OspF from Shigella flexneri, uncovering an intrinsic preference for p38 and Erk MAP kinase activation loops and revealing the enzyme residues that influence its selectivity for phosphothreonine. Our results establish a general method for linking phosphorylation sites to the enzymes that remove them, providing a means to dissect a key component of cellular regulatory networks.

biochemistry↗

bioRxiv2021

Auditory Hypersensitivity and Processing Deficits in a Rat Model of Fragile X Syndrome

Fragile X (FX) syndrome is one of the leading inherited causes of autism spectrum disorder (ASD). A majority of FX and ASD patients exhibit sensory hypersensitivity, including auditory hypersensitivity or hyperacusis, a condition in which everyday sounds are perceived as much louder than normal. Auditory processing deficits in FX and ASD also afford the opportunity to develop objective and quantifiable outcome measures that are likely to translate between humans and animal models due to the well-conserved nature of the auditory system and well-developed behavioral read-outs of sound perception. Therefore, in this study we characterized auditory hypersensitivity in a Fmr1 knockout (KO) transgenic rat model of FX using an operant conditioning task to assess sound detection thresholds and suprathreshold auditory reaction time-intensity (RT-I) functions, a reliable psychoacoustic measure of loudness growth, at a variety of stimulus frequencies, bandwidths and durations. Male Fmr1 KO and littermate WT rats both learned the task at the same rate and exhibited normal hearing thresholds. However, Fmr1 KO rats had faster auditory RTs over a broad range of intensities and steeper RT-I slopes than WT controls, perceptual evidence of excessive loudness growth in Fmr1 KO rats. Furthermore, we found that Fmr1 KO animals exhibited abnormal perceptual integration of sound duration and bandwidth, with diminished temporal but enhanced spectral integration of sound intensity. Because temporal and spectral integration of sound stimuli were altered in opposite directions in Fmr1 KO rats, this suggests that abnormal RTs in these animals are evidence of aberrant auditory processing rather than generalized hyperactivity or altered motor responses. Together, these results are indicative of fundamental changes to low-level auditory processing in Fmr1 KO animals. Finally, we demonstrated that antagonism of metabotropic glutamate receptor 5 (mGlu5) selectively and dose-dependently restored normal loudness growth in Fmr1 KO rats, suggesting a pharmacologic approach for alleviating sensory hypersensitivity associated with FX. This study leverages the tractable nature of the auditory system and the unique behavioral advantages of rats to provide important insights into the nature of a centrally important yet understudied aspect of FX and ASD.

neuroscience↗