The activation of lipases has been postulated to proceed by interfacial activation, temperature switch activation, or aqueous activation. Recently, based on molecular dynamics (MD) simulation experiments, the T1 lipase activation mechanism was proposed to involve aqueous activation in addition to a double-flap mechanism. Because the open conformation structure is still unavailable, it is difficult to validate the proposed theory unambiguously to understand the behavior of the enzyme. In this study, we try to validate the previous reports and uncover the mystery behind the activation process using structural analysis and MD simulations. To investigate the effects of temperature and environmental conditions on the activation process, MD simulations in different solvent environments (water and water-octane interface) and temperatures (20, 50, 70, 80, and 100°C) were performed. Based on the structural analysis of the lipases in the same family of T1 lipase (I.5 lipase family), we proposed that the lid domain comprises a6 and a7 helices connected by a loop, thus forming a helix-loop-helix motif involved in interfacial activation. Throughout the MD simulations experiments, lid displacements were only observed in the water-octane interface, not in the aqueous environment with respect to the temperature effect, suggesting that the activation process is governed by interfacial activation coupled with temperature switch activation. Examining the activation process in detail revealed that the large structural rearrangement of the lid domain was caused by the interaction between the hydrophobic residues of the lid with octane, a nonpolar solvent, and this conformation was found to be thermodynamically favorable.

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The ubiquitin ligase CRL4Cdt2 plays a vital role in preserving genomic integrity by regulating essential proteins during S phase and after DNA damage. Deregulation of CRL4Cdt2 during the cell cycle can cause DNA re-replication, which correlates with malignant transformation and tumor growth. CRL4Cdt2 regulates a broad spectrum of cell cycle substrates for ubiquitination and proteolysis, including Cdc10-dependent transcript 1 or Chromatin licensing and DNA replication factor 1 (Cdt1), histone H4K20 mono-methyltransferase (Set8) and cyclin-dependent kinase inhibitor 1 (p21), which regulate DNA replication. However, the mechanism it operates via its substrate receptor, Cdc10-dependent transcript 2 (Cdt2), is not fully understood. This review describes the essential features of the N-terminal and C-terminal parts of Cdt2 that regulate CRL4 ubiquitination activity, including the substrate recognition domain, intrinsically disordered region (IDR), phosphorylation sites, the PCNA-interacting protein-box (PIP) box motif and the DNA binding domain. Drugs targeting these specific domains of Cdt2 could have potential for the treatment of cancer.