Browsing by Subject "Polysome profiling"

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    Analysis of Translational Regulation Using Polysome Profiling and Puromycin Incorporation
    (Elsevier, 2025) Wood, Quinton W.; Mastracci, Teresa L.; Biology, School of Science
    Translation is the process of decoding an mRNA transcript to permit the synthesis of a protein. This process occurs in three steps: initiation, elongation, and termination. Each step of translation is regulated by translation factors. By regulating translation, the quantity and quality of proteins can be controlled. When translation becomes dysfunctional, disease can ensue, making translational regulation an important avenue of research. Polysome profiling and puromycin incorporation are experimental techniques used in concert to analyze the translational state of cells or tissues. Polysome profiling evaluates the state of translation by quantifying mRNAs based on the abundance of associated ribosomes. Puromycin incorporation measures the amount of newly synthesized protein. Together these methodologies can decipher stark and subtle changes in the rate and efficiency of translation, and provide the opportunity to dissect alterations to the translation of specific transcripts.
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    Autophagy-related 7 (ATG7) regulates food intake and liver health during asparaginase exposure
    (Elsevier, 2025) Zalma, Brian A.; Ibrahim, Maria; Rodriguez-Polanco, Flavio C.; Bhavsar, Chintan T.; Rodriguez, Esther M.; Cararo-Lopes, Eduardo; Farooq, Saad A.; Levy, Jordan L.; Wek, Ronald C.; White, Eileen; Anthony, Tracy G.; Biochemistry and Molecular Biology, School of Medicine
    Amino acid starvation by the chemotherapy agent asparaginase is a potent activator of the integrated stress response (ISR) in the liver and can upregulate autophagy in some cell types. We hypothesized that autophagy-related 7 (ATG7), a protein that is essential for autophagy and an ISR target gene, was necessary during exposure to asparaginase to maintain liver health. We knocked down Atg7 systemically (Atg7Δ/Δ) or in hepatocytes only (ls-Atg7KO) in mice before exposure to pegylated asparaginase for 5 days. Intact mice injected with asparaginase lost body weight due to reduced food intake and increased energy expenditure. Systemic Atg7 ablation reduced liver protein synthesis and increased liver injury in vehicle-injected mice but did not further reduce liver protein synthesis, exacerbate steatosis or liver injury, or alter energy expenditure following 5 days of asparaginase exposure. Atg7Δ/Δ mice were unexpectantly protected from asparaginase-induced anorexia and weight loss. This protection corresponded with reduced phosphorylation of hepatic GCN2 and blunted increases in ISR gene targets including growth differentiation factor 15 (GDF15), a negative regulator of food intake. Interestingly, asparaginase elevated serum GDF15 and reduced food intake in ls-Atg7KO mice, similar to intact mice. Liver triglycerides and production of the hepatokine fibroblast growth factor 21, another ISR gene target, were suppressed in asparaginase-exposed Atg7Δ/Δ and ls-Atg7KO mice. This work identifies a bidirectional relationship between autophagy and the ISR in the liver during asparaginase, affecting food intake and liver health.