Catalytically active holoHomo sapiensadenosine deaminase I adopts a closed conformation

Abstract

Homo sapiens adenosine deaminase 1 (HsADA1; UniProt P00813) is an immunologically relevant enzyme with roles in T-cell activation and modulation of adenosine metabolism and signaling. Patients with genetic deficiency in HsADA1 suffer from severe combined immunodeficiency, and HsADA1 is a therapeutic target in hairy cell leukemias. Historically, insights into the catalytic mechanism and the structural attributes of HsADA1 have been derived from studies of its homologs from Bos taurus (BtADA) and Mus musculus (MmADA). Here, the structure of holo HsADA1 is presented, as well as biochemical characterization that confirms its high activity and shows that it is active across a broad pH range. Structurally, holo HsADA1 adopts a closed conformation distinct from the open conformation of holo BtADA. Comparison of holo HsADA1 and MmADA reveals that MmADA also adopts a closed conformation. These findings challenge previous assumptions gleaned from BtADA regarding the conformation of HsADA1 that may be relevant to its immunological interactions, particularly its ability to bind adenosine receptors. From a broader perspective, the structural analysis of HsADA1 presents a cautionary tale for reliance on homologs to make structural inferences relevant to applications such as protein engineering or drug development.

Engineering CREB‐activated promoters for adenosine‐induced gene expression

“…[46][47][48][49] Typical heat-deactivation protocols performed by FBS suppliers (30 min at 56 • C) do not eliminate this ADO degradation activity, potentially due to the higher than 56 • C melting temperature of mammalian ADO deaminase enzymes. [50] ADO and CREB-signaling are highly implicated in immune suppression of T cells in tumor environments and have been shown to limit T cell function via A 2A R. [51,52] We have shown that our synthetic promoters are strongly induced at physiologically relevant levels of ADO, that is, at levels of ADO found in solid tumors, and that they have a low off-state expression level, that is, without exposure to ADO. We have also shown that it is possible to utilize an ADO-activated synthetic promoter to drive cell-based, potentially tumor-localized expression of agents (e.g., IL-2) that have antitumor activity but are toxic when present systemically.…”

“…[ 46–49 ] Typical heat‐deactivation protocols performed by FBS suppliers (30 min at 56°C) do not eliminate this ADO degradation activity, potentially due to the higher than 56°C melting temperature of mammalian ADO deaminase enzymes. [ 50 ]…”