Proteomic analysis reveals novel ligands and substrates for LNX1 E3 ubiquitin ligase

Lenihan, Joan A.; Saha, Orthis; Young, Paul

doi:10.1371/journal.pone.0187352

Cited by 10 publications

(20 citation statements)

References 36 publications

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“…This suggests the possibility of differential binding of LNX1 and LNX2 to certain ligands. Indeed this was subsequently shown in proteomics studies in which the preferential binding of LNX1 PDZ2 to ligands with carboxyl terminal Cysteine residues in particular was noted [13,14,18]. Such LNX1-or LNX2-specific interactions may underlie specialized functions of LNX1/2 paralogues in vertebrates.…”

Section: Pdz Domainsmentioning

confidence: 78%

“…The motions induced within the PDZ domain by the electric-field were postulated to reflect the conformational changes associated with ligand binding. Further analysis of these motions may provide insights into the mechanism of ligand binding by this PDZ domain and through comparison with the LNX1 PDZ2 structure may elucidate the structural basis for the differential binding of certain ligands to PDZ2 from LNX1 compared with LNX2 [13,14].…”

Section: Pdz Domainsmentioning

confidence: 99%

“…Since they lack catalytic activity but retain the NPAY motif and PDZ protein interaction domains, these neuronally expressed isoforms might be expected to have opposite effects from LNX1p80 or LNX2 towards substrate proteins-interacting with but not ubiquitinating them, and perhaps protecting them from ubiquitination by LNX2 in situations in which they are co-expressed. However, in some cases it appears that LNX1p70, despite lacking a catalytic domain, can nevertheless promote the ubiquitination of certain interacting proteins (LIPRIN-α1 and KLHL11), most likely by acting as a scaffold to recruit other ubiquitin ligases [14]. In addition, the neuronal LNX1 isoforms may have scaffold functions that are not related to ubiquitination.…”

Section: Lnx1/2 Protein Expressionmentioning

confidence: 99%

“…Clustering analysis for LNX1 interacting proteins based on gene ontology annotations have provided some insights into potentially novel LNX1 functions [9,18]. More recently affinity purification combined with MS has been employed to characterize the LNX1 interactome in the HEK293 cell line and LNX1 and LNX2 PDZ2 interacting proteins from mouse brain lysates [13,14]. Given the inherent limitations of these high throughput approaches I focus below on those interactions that have at least been verified using full-length proteins expressed in mammalian cells (Table 1).…”

Section: Interactions Of Lnx1/2 Proteins With Ligands Other Than Numbmentioning

confidence: 99%

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LNX1/LNX2 proteins: functions in neuronal signalling and beyond

Young

2018

Neuronal Signaling

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Ligand of NUMB Protein X1 and X2 (LNX1 and LNX2) are E3 ubiquitin ligases, named for their ability to interact with and promote the degradation of the cell fate determinant protein NUMB. On this basis they are thought to play a role in modulating NUMB/NOTCH signalling during processes such as cortical neurogenesis. However, LNX1/2 proteins can bind, via their four PDZ (PSD95, DLGA, ZO-1) domains, to an extraordinarily large number of other proteins besides NUMB. Many of these interactions suggest additional roles for LNX1/2 proteins in the nervous system in areas such as synapse formation, neurotransmission and regulating neuroglial function. Twenty years on from their initial discovery, I discuss here the putative neuronal functions of LNX1/2 proteins in light of the anxiety-related phenotype of double knockout mice lacking LNX1 and LNX2 in the central nervous system (CNS). I also review what is known about non-neuronal roles of LNX1/2 proteins, including their roles in embryonic patterning and pancreas development in zebrafish and their possible involvement in colorectal cancer (CRC), osteoclast differentiation and immune function in mammals. The emerging picture places LNX1/2 proteins as potential regulators of multiple cellular signalling processes, but in many cases the physiological significance of such roles remains only partly validated and needs to be considered in the context of the tight control of LNX1/2 protein levels in vivo.

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Section: Pdz Domainsmentioning

confidence: 78%

Section: Pdz Domainsmentioning

confidence: 99%

Section: Lnx1/2 Protein Expressionmentioning

confidence: 99%

Section: Interactions Of Lnx1/2 Proteins With Ligands Other Than Numbmentioning

confidence: 99%

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LNX1/LNX2 proteins: functions in neuronal signalling and beyond

Young

2018

Neuronal Signaling

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“…They share identical domain structure comprised of one amino‐terminal RING domain and four PDZ domains (Rice, Northcutt, & Kurschner, ). Each of the four PDZ domains of LNX1 and LNX2 interact with specific classes of PDZ ligands (Lenihan, Saha, & Young, ; Wolting et al., ). LNX2 is expressed in many tissues, including adult brain (Lenihan, Saha, Mansfield, & Young, ; Rice et al., ).…”

Section: Introductionmentioning

confidence: 99%

E3 ubiquitin ligases LNX1 and LNX2 localize at neuronal gap junctions formed by connexin36 in rodent brain and molecularly interact with connexin36

Lynn

Hormuzdi

et al. 2018

Eur J of Neuroscience

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Electrical synapses in the mammalian central nervous system (CNS) are increasingly recognized as highly complex structures for mediation of neuronal communication, both with respect to their capacity for dynamic short‐ and long‐term modification in efficacy of synaptic transmission and their multimolecular regulatory and structural components. These two characteristics are inextricably linked, such that understanding of mechanisms that contribute to electrical synaptic plasticity requires knowledge of the molecular composition of electrical synapses and the functions of proteins associated with these synapses. Here, we provide evidence that the key component of gap junctions that form the majority of electrical synapses in the mammalian CNS, namely connexin36 (Cx36), directly interacts with the related E3 ubiquitin ligase proteins Ligand of NUMB protein X1 (LNX1) and Ligand of NUMB protein X2 (LNX2). This is based on immunofluorescence colocalization of LNX1 and LNX2 with Cx36‐containing gap junctions in adult mouse brain versus lack of such coassociation in LNX null mice, coimmunoprecipitation of LNX proteins with Cx36, and pull‐down of Cx36 with the second PDZ domain of LNX1 and LNX2. Furthermore, cotransfection of cultured cells with Cx36 and E3 ubiquitin ligase‐competent LNX1 and LNX2 isoforms led to loss of Cx36‐containing gap junctions between cells, whereas these junctions persisted following transfection with isoforms of these proteins that lack ligase activity. Our results suggest that a LNX protein mediates ubiquitination of Cx36 at neuronal gap junctions, with consequent Cx36 internalization, and may thereby contribute to intracellular mechanisms that govern the recently identified modifiability of synaptic transmission at electrical synapses.

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E3 ubiquitin ligases LNX1 and LNX2 are major regulators of the presynaptic glycine transporter GlyT2

Rocha-Muñoz

Núñez

Arribas-González

et al. 2019

Sci Rep

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The neuronal glycine transporter GlyT2 is an essential regulator of glycinergic neurotransmission that recaptures glycine in presynaptic terminals to facilitate transmitter packaging in synaptic vesicles. Alterations in GlyT2 expression or activity result in lower cytosolic glycine levels, emptying glycinergic synaptic vesicles and impairing neurotransmission. Lack of glycinergic neurotransmission caused by GlyT2 loss-of-function mutations results in Hyperekplexia, a rare neurological disease characterized by generalized stiffness and motor alterations that may cause sudden infant death. Although the importance of GlyT2 in pathology is known, how this transporter is regulated at the molecular level is poorly understood, limiting current therapeutic strategies. Guided by an unbiased screening, we discovered that E3 ubiquitin ligase Ligand of Numb proteins X1/2 (LNX1/2) modulate the ubiquitination status of GlyT2. The N-terminal RING-finger domain of LNX1/2 ubiquitinates a cytoplasmic C-terminal lysine cluster in GlyT2 (K751, K773, K787 and K791), and this process regulates the expression levels and transport activity of GlyT2. The genetic deletion of endogenous LNX2 in spinal cord primary neurons causes an increase in GlyT2 expression and we find that LNX2 is required for PKC-mediated control of GlyT2 transport. This work identifies, to our knowledge, the first E3 ubiquitin-ligases acting on GlyT2, revealing a novel molecular mechanism that controls presynaptic glycine availability. Providing a better understanding of the molecular regulation of GlyT2 may help future investigations into the molecular basis of human disease states caused by dysfunctional glycinergic neurotransmission, such as hyperekplexia and chronic pain.

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Proteomic analysis reveals novel ligands and substrates for LNX1 E3 ubiquitin ligase

Cited by 10 publications

References 36 publications

LNX1/LNX2 proteins: functions in neuronal signalling and beyond

LNX1/LNX2 proteins: functions in neuronal signalling and beyond

E3 ubiquitin ligases LNX1 and LNX2 localize at neuronal gap junctions formed by connexin36 in rodent brain and molecularly interact with connexin36

E3 ubiquitin ligases LNX1 and LNX2 are major regulators of the presynaptic glycine transporter GlyT2

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