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The tuberous sclerosis protein complex (TSCC) is a master negative regulator of the mTORC1 signaling pathway through hydrolyzing the GTP loaded on the small GTPase Rheb, which is a key activator of mTOR. However, the large size (~700\u2009kDa) and complex structural organization of TSCC render it vulnerable to degradation and inactivation, thus limiting its potential application. In this work, based on thorough analysis and understanding of the structural mechanism of how the stabilization domain of TSC2 secures the association of TSC2\u2010GAP with Rheb and thus enhances its GAP activity, we designed two proteins, namely SSG\u2010MTM (short stabilization domain and GAP domain\u2010membrane targeting motif) and SSG\u2010TSC1N, which were able to function like TSCC to negatively regulate Rheb and mTORC1, but with much\u2010reduced sizes (~1\/15 and\u2009~\u20091\/9 of the size of TSCC, respectively). Biochemical and cell biological assays demonstrated that these designed proteins indeed could promote the GTPase activity of Rheb to hydrolyze GTP, inhibit the kinase activity of mTORC1, and prevent mTORC1 from down\u2010regulating catabolism and autophagy.<\/jats:p>","DOI":"10.1002\/pro.4731","type":"journal-article","created":{"date-parts":[[2023,7,18]],"date-time":"2023-07-18T15:36:01Z","timestamp":1689694561000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Design of negative\u2010regulating proteins of Rheb\/<scp>mTORC1<\/scp>with much\u2010reduced sizes of the tuberous sclerosis protein complex"],"prefix":"10.1002","volume":"32","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9148-9168","authenticated-orcid":false,"given":"Wencheng","family":"Fu","sequence":"first","affiliation":[{"name":"State Key Laboratory of Microbial Metabolism, School of Life Sciences &amp; Biotechnology, the Joint International Research Laboratory of Metabolic &amp; Developmental Sciences MOE Shanghai Jiao Tong University Shanghai China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Geng","family":"Wu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Microbial Metabolism, School of Life Sciences &amp; Biotechnology, the Joint International Research Laboratory of Metabolic &amp; Developmental Sciences MOE Shanghai Jiao Tong University Shanghai China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2023,7,29]]},"reference":[{"key":"e_1_2_8_2_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cmet.2009.11.010"},{"key":"e_1_2_8_3_1","first-page":"1201","article-title":"Tuberous sclerosis\u2010associated renal cell carcinoma. 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