Functional characterisation of LNX1 and LNX2 proteins

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Lenihan, Joan A.
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University College Cork
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Ligand of Numb protein X1 (LNX1) and LNX2 are E3 ubiquitin ligases that contain a catalytic RING (Really Interesting New Gene) domain and four PDZ (PSD-95, DlgA, ZO-1) domains. LNX1 and LNX2 can interact with Numb – a key regulator of neurogenesis and neuronal differentiation. LNX1 can target Numb for proteasomal degradation, and Lnx mRNAs are prominently expressed in the nervous system, suggesting that LNX proteins play a role in neural development. This hypothesis remains unproven, however, and our understanding of LNX protein function is very limited – largely because LNX proteins are present at very low levels in vivo. Chapter 2 of this thesis addresses this – investigating possible reasons for the low levels of LNX proteins observed in vivo, at both transcriptional and translational levels, and also in terms of protein stability. Luciferase reporter assays show that the 5’ untranslated region of the Lnx1_variant 2 mRNA, that generates the LNX1p70 isoform, strongly suppresses protein production. This effect is mediated in part by the presence of upstream open reading frames (uORFs), but also by a sequence element that decreases both mRNA levels and translational efficiency. By contrast, uORFs do not negatively regulate LNX1p80 or LNX2 expression. Instead, some evidence is presented that protein turnover via proteasomal degradation may influence LNX1p80 levels in cells. To gain functional insights into the LNX family, Chapter 3 details the first physiologically relevant affinity purification/mass spectrometry-based analysis of the LNX interactome. In the context of mammalian cells, this approach identified a large number of novel LNX1-interacting proteins, as well as confirming known interactions with NUMB and ERC2. Many of the novel interactions mapped to the LNX PDZ domains, particularly PDZ2, and many showed specificity for LNX1 over the closely related LNX2. It was shown that LIPRIN-α1, KLHL11, KIF7 and ERC2 are substrates for ubiquitination by LNX1. LNX1 ubiquitination of LIPRIN-α1 is dependent on a PDZ binding motif containing a carboxyl terminal cysteine that binds LNX1 PDZ2. Surprisingly, the neuronally-expressed LNX1p70 isoform, that lacks the RING domain, was found to promote ubiquitination of Liprin-α1 and KLHL11, albeit to a lesser extent than the longer RING-containing LNX1p80 isoform. Of several E3-ligases identified in the LNX1 interactome, interactions of LNX1 with MID2/TRIM1 and TRIM27 were confirmed. On this basis, a model is proposed, whereby LNX1p70 - despite lacking a catalytic RING domain, may function as a scaffold to promote ubiquitination of its ligands through recruitment of other E3-ligases. Proteomic analysis of LNX-interacting proteins in the context of brain tissue identified and/or confirmed interactions of LNX1 and LNX2 with proteins known to have presynaptic and neuronal signalling functions, including the presynaptic active zone constituents ERC1, ERC2, and LIPRIN-s (PPFIA1, PPFIA3), as well as the F-BAR domain proteins FCHSD2 (nervous wreck homolog) and SRGAP2. To examine the role of LNX proteins in vivo, mice lacking both LNX1 and LNX2 expression in the brain were generated. Surprisingly, these mice are viable, fertile and physically healthy (Chapter 4). Behavioural analysis of LNX1/LNX2 double knockout mice revealed decreased anxiety-related behaviour, as assessed in the open field and elevated plus maze paradigms. By contrast, no major defects in learning, motor or sensory function were observed. The proteomic analysis (Chapter 3) revealed several novel neuronal LNX-interacting protein candidates that might contribute to the anxiolytic phenotype observed. Overall, these findings provide novel functional insights into the LNX protein family and identify promising candidates to mediate LNX functions in the central nervous system.
Lenihan, J. A. 2022. Functional characterisation of LNX1 and LNX2 proteins. PhD Thesis, University College Cork.