Intrinsic Bias of the Genetic Code Shapes the Folding and Stability Landscapes of Microproteins. Guo et al
Description
This dataset contains the raw immunofluorescence microscopy images, uncropped Western blot data, predicted protein structures, and analysis code corresponding to all experiments and analyses presented in the manuscript. The immunofluorescence microscopy images were generated to investigate the intracellular stability and degradation pathways of microproteins translated from regulatory upstream open reading frames (uORFs). The working hypothesis is that microproteins encoded by these regulatory uORFs are selectively degraded through cellular protein quality control pathways, including the ubiquitin–proteasome system or the autophagy–lysosome pathway. To test this hypothesis, cells expressing HA-tagged microproteins derived from regulatory uORFs were treated with the proteasome inhibitor MG132 or the lysosome inhibitor Chloroquine (CQ). These pharmacological perturbations were used to determine whether inhibition of specific degradation pathways results in the accumulation of the microproteins. After immunofluorescence staining, images were acquired using an Olympus FV3000 laser scanning confocal microscope equipped with a 60 × oil immersion objective. Z-stack images were collected with a step size of 0.7 μm. Raw fluorescence images were processed and quantified using ImageJ. The data can be used to compare fluorescence intensity changes between different treatment conditions and to evaluate the stability of microproteins under perturbation of protein degradation pathways. The results indicate that microproteins encoded by these regulatory uORFs are primarily degraded through the ubiquitin–proteasome system, as evidenced by their accumulation following treatment with the proteasome inhibitor MG132, whereas inhibition of lysosomal degradation using CQ shows comparatively limited effects. The second part of the dataset provides the uncropped source data for all Western blot experiments presented in the manuscript. These images correspond to the Western blot figures shown in both the main figures and supplementary figures and are provided to ensure transparency and allow independent assessment and verification of the reported results. The third part of the dataset contains predicted protein structures of all microproteins analyzed in this study, organized within a single directory. Two compressed archives are provided, corresponding to structures generated using ESMFold and AlphaFold2, respectively. The fourth part of the dataset provides the code used in this study, including scripts and notebooks for structural analysis and ESM-2–based modeling of microprotein stability, supporting data processing, statistical analysis, and figure generation.
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Institutions
- Zhejiang UniversityZhejiang, Hangzhou