Proteolytic stability and aggregation in a key metabolic enzyme of bacteria

• 成果类型: Article
• 署名作者: Pollack, Dan; Nozoe, Takashi; Kussell, Edo
• 署名单位: New York University; University of Tokyo; University of Tokyo; University of Tokyo; New York University
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-10026
• DOI: 10.1073/pnas.2301458121
• 发表日期: 2024-05-07
• 关键词: galactosidase alpha-complementation; lacz beta-galactosidase; ssra degradation tag; amino-acid-sequence; escherichia-coli; protein aggregation; peptide; expression; SYSTEM; gene

摘要

Proteins that are kinetically stable are thought to be less prone to both aggregation and proteolysis. We demonstrate that the classical lac system of Escherichia coli can be leveraged as a model system to study this relation. beta- galactosidase (LacZ) plays a critical role in lactose metabolism and is an extremely stable protein that can persist in growing cells for multiple generations after expression has stopped. By attaching degradation tags to the LacZ protein, we find that LacZ can be transiently degraded during lac operon expression but once expression has stopped functional LacZ is protected from degradation. We reversibly destabilize its tetrameric assembly using alpha- complementation, and show that unassembled LacZ monomers and dimers can either be degraded or lead to formation of aggregates within cells, while the tetrameric state protects against proteolysis and aggregation. We show that the presence of aggregates is associated with cell death, and that these proteotoxic stress phenotypes can be alleviated by attaching an ssrA tag to LacZ monomers which leads to their degradation. We unify our findings using a biophysical model that enables the interplay of protein assembly, degradation, and aggregation to be studied quantitatively in vivo. This work may yield approaches to reversing and preventing protein - misfolding disease states, while elucidating the functions of proteolytic stability in constant and fluctuating environments.