The LNX Family of Multi-PDZ E3 Ligases: Using a Mutagenesis-based Approach to Establish the Role of PDZ Domains in LNX1 Function

LNX1 belongs to a family of multi-PDZ domain containing RING-type E3 ligases. Several interactions have been mapped to its PDZ domains, but the role of each domain in LNX function has not yet been determined. To study individual PDZ domain function in the context of full length protein I generated point mutations in peptide binding sites of each of PDZ domain, and in a putative phosphoinositide binding site of LNX1 PDZ4. Peptide binding was successfully disrupted by an arginine or lysine to alanine mutation in the peptide binding cleft. A LNX1 PDZ4 mutant with lysine residues in a putative phosphoinositide binding site mutated to glutamate displayed decreased membrane localization. The impact of each PDZ mutation on cell morphology and substrate ubiquitination was also investigated. I identified a potential role for PDZ binding in auto-inhibition of RING function. Additionally, novel interactions between LNX1 and Frizzled family members were identified and characterized.MAS

Biochemical and Computational Analysis Of LNX1 Interacting Proteins

PDZ (Post-synaptic density, 95 kDa, Discs large, Zona Occludens-1) domains are protein interaction domains that bind to the carboxy-terminal amino acids of binding partners, heterodimerize with other PDZ domains, and also bind phosphoinositides. PDZ domain containing proteins are frequently involved in the assembly of multi-protein complexes and clustering of transmembrane proteins. LNX1 (Ligand of Numb, protein X 1) is a RING (Really Interesting New Gene) domain-containing E3 ubiquitin ligase that also includes four PDZ domains suggesting it functions as a scaffold for a multiprotein complex. Here we use a human protein array to identify direct LNX1 PDZ domain binding partners. Screening of 8,000 human proteins with isolated PDZ domains identified 53 potential LNX1 binding partners. We combined this set with LNX1 interacting proteins identified by other methods to assemble a list of 220 LNX1 interacting proteins. Bioinformatic analysis of this protein list was used to select interactions of interest for future studies. Using this approach we identify and confirm six novel LNX1 binding partners: KCNA4, PAK6, PLEKHG5, PKC-alpha1, TYK2 and PBK, and suggest that LNX

Drug Toxicity Deaths after Release from Incarceration in Ontario, 2006-2013: Review of Coroner’s Cases

<div><p>Background</p><p>There is an increased risk of death due to drug toxicity after release from incarceration. The purpose of this study was to describe the timing, rate and circumstances of drug toxicity deaths following release from incarceration. This information can be used to help design potential preventive interventions.</p><p>Methods and Findings</p><p>We reviewed coroner’s files to identify deaths in adults in Ontario between 2006 and 2013 caused by drug toxicity (n = 6,978) and these records were matched with provincial correctional records to identify individuals who died within one year of being released from incarceration (n = 702). Twenty percent (n = 137) of the 702 deaths occurred within one week of release. The majority (77%, n = 538) of deaths after release involved one or more opioids. Of the deaths involving opioids, intervention by another person may have been possible in 318 cases.</p><p>Conclusions</p><p>Between 2006 and 2013 in Ontario, one in ten drug toxicity deaths in adults occurred within one year of release from provincial incarceration. These findings may help to inform the implemention and assessment of interventions aimed at reducing drug toxicity deaths following release from incarceration.</p></div

Number of deaths by week since release in the year after release from provincial incarceration, 2006–2013.

<p>Number of deaths by week since release in the year after release from provincial incarceration, 2006–2013.</p

Characteristics of persons who died due to drug intoxication in the year after release from incarceration in Ontario, 2006–2013, n = 702.

<p>Characteristics of persons who died due to drug intoxication in the year after release from incarceration in Ontario, 2006–2013, n = 702.</p

Flow diagram of study participants.

<p>Flow diagram of study participants.</p

Expected and observed mean annual deaths for persons who died due to drug intoxication in the year after release from incarceration in Ontario, 2006–2013.

<p>Expected and observed mean annual deaths for persons who died due to drug intoxication in the year after release from incarceration in Ontario, 2006–2013.</p

Number of deaths by day since release in the year after release from provincial incarceration, up to day 60, 2006–2013.

<p>Number of deaths by day since release in the year after release from provincial incarceration, up to day 60, 2006–2013.</p