K29-Linked Ubiquitin Signaling Regulates Proteotoxic Stress Response and Cell Cycle

Abstract

Protein ubiquitination, one of the most common posttranslational modifications, is involved in numerous cellular processes. It shows remarkable topological and functional diversity, a phenomenon known as the ubiquitin code, through the polymerization of ubiquitin via different linkages. Deciphering the cellular ubiquitin code is of central importance to understanding the physiology of the cell. Among the eight possible linkages, K29-linked polyubiquitin is a relatively abundant type of polyubiquitin in both human and yeast cells (Tsuchiya et al., 2018). Despite previous studies, the lack of specific binders as tools to detect K29-linked polyubiquitin has prevented further understanding of its function. In this study, we screened and characterized a synthetic antigen-binding fragment (named sAB-K29) that can specifically recognize K29-linked polyubiquitin. We determined the crystal structure of sAB-K29 bound to K29-linked diubiquitin (diUb), which revealed the molecular basis of sAB-K29 specificity. Using sAB-K29 as a tool, we identified the involvement of K29-linked ubiquitination in RNA processing, the proteotoxic stress response and cell cycle regulation. In particular, we showed that K29-linked ubiquitination is enriched in the midbody at the telophase of mitosis and that downregulation of the K29-linked ubiquitination signal arrests cells in G1/S phase.

The E3 ubiquitin ligase HECTD1 contributes to cell cycle progression through an effect on mitosis

“…This data which implicates K29-linked ubiquitylation during cell cycle progression, also revealed the presence of K29 linkages in mitotic cells, specifically at the midbody during telophase. In line with our data, HECTD1 and BUB3 were identified from pulldown and proteomics analysis using the K29-specific affimer [50]. This exciting new reagent now enables further studies to explore and the function of ubiquitin chains containing K29 linkages in various cellular context including during cell cycle progression.…”

“…In this study we explored HECTD1 function in the context of cell proliferation and found that loss of HECTD1 ubiquitin ligase activity reduces cell proliferation through an effect on mitosis. Together, our data support recent evidence implicating K29-linked ubiquitylation in mitotic regulation and provide novel insights into the 'ubiquitin code' [50].…”

“…Using TRABID NZF ubiquitin binding domain as bait, we provide proof-of-principle that ubiquitin chains likely containing K29-linkages can be trapped in different cell cycle stages. Although affimers specific for K33 and K6 ubiquitin linkages have been available for a few years, it is only during the preparation of this manuscript that a K29-specific affimer was reported [50]. Excitingly this study revealed the enrichment of K29-linked ubiquitin in stress response and cell cycle regulation.…”

K27‐linked ubiquitylation promotes p97 substrate processing and is essential for cell proliferation

“…Where indicated, cells were pre‐extracted prior to fixation using either CSK buffer (100 mM NaCl; 10 mM HEPES; 3 mM MgCl 2 ; 300 mM sucrose; 0.25% Triton‐X; 1 mM PMSF) or a stringent pre‐extraction buffer (10 mM Tris‐HCl, pH 7.4; 2.5 mM MgCl 2 ; 0.5% igepal; 1 mM PMSF). K29‐linked Ub conjugates were visualized using a K29‐Ub‐specific synthetic antigen binder (sAB‐K29; kind gift from Minglei Zhao (University of Chicago)) (Yu et al , 2021) and Alexa Fluor 488 AffiniPure F(ab′)2 Fragment Donkey Anti‐Human IgG (Jackson Immunoresearch). Local UV damage was induced by irradiating cells on coverslips with 100 J/m 2 through a PBS‐equilibrated membrane of 8 μm pores and fixing at the indicated timepoints.…”

Ubiquitination in T-Cell Activation and Checkpoint Inhibition: New Avenues for Targeted Cancer Immunotherapy

“…Lys27-linked chains appear important for regulating innate immunity [ 41 ], cell cycle [ 42 ], and mitochondrial functions [ 43 ]. Finally, Lys6-chains have been described to participate in DNA repair [ 44 ] and mitophagy [ 45 ], whereas, Lys29-chains have been associated with Wnt signaling [ 46 ], and just recently, proteotoxic stress and cell cycle [ 47 ]. Lys33-chains play a crucial role in post-Golgi trafficking [ 48 ] and T-cell receptor (TCR) regulation [ 49 , 50 ].…”