Biology subjects

Lewis, P.

Publications and source records attributed to Lewis, P..

23 records · Page 2Linked to original sources

bioRxiv2020

Sialylation of Asparagine 612 inhibits Aconitase activity during mouse sperm capacitation; A possible mechanism for the switch from oxidative phosphorylation to glycolysis

After ejaculation, mammalian spermatozoa must undergo a process known as capacitation in order to successfully fertilize the oocyte. Several post-translational modifications occur during capacitation, including sialylation, which despite being limited to a few proteins, seems to be essential for proper sperm-oocyte interaction. Regardless of its importance, to date, no single study has ever identified nor quantified which glycoproteins bearing terminal sialic acid (Sia) are altered during capacitation. Here we characterize sialylation during mouse sperm capacitation. Using tandem mass spectrometry coupled with liquid chromatography (LC-MS/MS), we found 142 non-reductant peptides, with 9 of them showing potential modifications on their sialylated oligosaccharides during capacitation. As such, N-linked sialoglycopeptides from C4b-binding protein, endothelial lipase (EL), serine proteases 39 and 52, testis-expressed protein 101 and zonadhesin were reduced following capacitation. In contrast, mitochondrial aconitate hydratase (aconitase; ACO2) was the only protein to show an increase in Sia content during capacitation. Interestingly, while the loss of Sia within EL (N62) was accompanied by a reduction in its phospholipase A1 activity, the increase of sialylation in the ACO2 (N612) also resulted in a decrease of the activity of this TCA cycle enzyme. The latter was confirmed by N612D recombinant protein with both His and GFP tag, in which the N612D mutant had no activity compared to WT when protein. Computer modelling show that N612 sits atop the catalytic site of ACO2. The introduction of sialic acid causes a large confirmation change in the alpha helix, essentially, distorting the active site, leading to complete loss of function. These findings suggest that the switch from oxidative phosphorylation, over to glycolysis that occurs during capacitation may come about through sialylation of ACO2.

developmental biology↗

bioRxiv2020

Sorting nexin-27 regulates AMPA receptor trafficking through the synaptic adhesion protein LRFN2

The endosome-associated cargo adaptor sorting nexin-27 (SNX27) is linked to various neuropathologies through sorting of integral proteins to the synaptic surface, most notably AMPA receptors. To provide a broader view of SNX27-associated pathologies we have performed unbiased proteomics to identify new neuronal SNX27-dependent cargoes, and identified proteins linked to excitotoxicity (SLC1A3, SLC4A7, SLC6A11), epilepsy, intellectual disabilities and working memory deficits (KCNT2, ADAM22, KIDINS220, LRFN2). Focusing on the synaptic adhesion molecule leucine-rich repeat and fibronectin type-III domain-containing protein 2 (LRFN2), we establish that SNX27 binds to LRFN2 and is responsible for regulating its endosomal sorting. LRFN2 associates with AMPA receptors and knockdown of LRFN2 phenocopies SNX27 depletion in decreasing surface expression of AMPA receptors, reducing synaptic activity and attenuating hippocampal long-term potentiation. Our evidence suggests that, in contrast to previous reports, SNX27 does not directly bind to AMPA receptors, and instead controls AMPA receptor-mediated synaptic transmission and plasticity indirectly through the endosomal sorting of LRFN2. Overall, our study provides new molecular insight into the perturbed function of SNX27 and LRFN2 in a range of neurological conditions.

molecular biology↗

bioRxiv2020

Regulation of mitophagy by the NSL complex underlies genetic risk for Parkinson's disease at Chr16q11.2 and on the MAPT H1 allele.

Parkinsons disease (PD) is a common incurable neurodegenerative disease. The identification of genetic variants via genome-wide association studies (GWAS) has considerably advanced our understanding of the PD genetic risk. Understanding the functional significance of the risk loci is now a critical step towards translating these genetic advances into an enhanced biological understanding of the disease. Impaired mitophagy is a key causative pathway in familial PD, but its relevance to idiopathic PD is unclear. We used a mitophagy screening assay to evaluate the functional significance of risk genes identified through GWAS. We identified two new regulators of PINK1-mitophagy, KAT8 and KANSL1, previously shown to modulate lysine acetylation. We show that KAT8 and KANSL1 modulate PINK1 gene expression and subsequent PINK1-mitophagy. These findings suggest PINK1-mitophagy is a contributing factor to idiopathic PD. KANSL1 is located on chromosome 17q21 where the risk associated gene has long been considered to be MAPT. While our data does not exclude a possible association between the MAPT gene and PD, it provides strong evidence that KANSL1 plays a crucial role in the disease. Finally, these results enrich our understanding of physiological events regulating mitophagy and establish a novel pathway for drug targeting in neurodegeneration.

neuroscience↗

bioRxiv2018

Assessing Combinability of Phylogenomic Data using Bayes Factors

With the rapid reduction in sequencing costs of high-throughput genomic data, it has become commonplace to use hundreds of genes/sites to infer phylogeny of any study system. While sampling large number of genes has given us a tremendous opportunity to uncover previously unknown relationships and improve phylogenetic resolution, it also presents us with new challenges when the phylogenetic signal is confused by differences in the evolutionary histories of sampled genes. Given the addition of accurate marginal likelihood estimation methods into popular Bayesian software programs, it is natural to consider using the Bayes Factor (BF) to compare different partition models in which genes within any given partition subset share both tree topology and edge lengths. We explore using marginal likelihood to assess data subset combinability when data subsets have varying levels of phylogenetic discordance due to deep coalescence events among genes (simulated within a species tree), and compare the results with our recently-described phylogenetic informational dissonance index (D) estimated for each data set. BF effectively detects phylogenetic incongruence, and provides a way to assess the statistical significance of D values. We discuss methods for calibrating BFs, and use calibrated BFs to assess data combinability using an empirical data set comprising 56 plastid genes from green algae order Volvocales.

evolutionary biology↗

bioRxiv2017

ATRX, DAXX or MEN1 mutant pancreatic neuroendocrine tumors are a distinct alpha-cell signature subgroup

The most commonly mutated genes in pancreatic neuroendocrine tumors (PanNETs) are ATRX, DAXX, and MEN1. Little is known about the cells-of-origin for non-functional neuroendocrine tumors. Here, we genotyped 64 PanNETs for mutations in ATRX, DAXX, and MEN1 and found 37 tumors (58%) carry mutations in these three genes (A-D-M mutant PanNETs) and this correlates with a worse clinical outcome than tumors carrying the wild-type alleles of all three genes (A-D-M WT PanNETs). We performed RNA sequencing and DNA-methylation analysis on 33 randomly selected cases to reveal two distinct subgroups with one group consisting entirely of A-D-M mutant PanNETs. Two biomarkers differentiating A-D-M mutant from A-D-M WT PanNETs were high ARX gene expression and low PDX1 gene expression with PDX1 promoter hyper-methylation in the A-D-M mutant PanNETs. Moreover, A-D-M mutant PanNETs had a gene expression signature related to that of alpha cells (pval < 0.009) of pancreatic islets including increased expression of HNF1A and its transcriptional target genes. This gene expression profile suggests that A-D-M mutant PanNETs originate from or transdifferentiate into a distinct cell type similar to alpha cells.

cancer biology↗