Biology subjects

James, A. M.

Publications and source records attributed to James, A. M..

5 recordsLinked to original sources

bioRxiv2026

CLN6 is a glycerophosphoglycerol epimerase in bis(monoacylglycero)phosphate biosynthesis

Lysosomal catabolism depends on bis(monoacylglycero)phosphate (BMP), a phospholipid with atypical S,S stereochemistry of unknown biosynthetic origin. CLN8 and CLN5 catalyze sequential acylation steps in BMP synthesis, and their loss of function causes Batten disease, a group of inherited neurodegenerative lysosomal storage disorders. Here we identify CLN6, a biochemically uncharacterized endoplasmic reticulum protein whose loss also causes Batten disease, as the calcium-dependent glycerophosphoglycerol epimerase that establishes BMP stereochemistry upstream of CLN8. CLN6 deficiency depletes BMP in cells, mice, and patient lysosomes, and reduces lysosomal hydrolase levels. BMP-precursor supplementation bypasses CLN6, restores BMP and increases hydrolase abundance in CLN6-deficient cells and mice. These findings place three Batten disease proteins in a single biosynthetic pathway and reveal BMP deficiency as a reversible cause of disrupted lysosomal hydrolase homeostasis.

biochemistry↗

bioRxiv2025

Stereospecific GPG acylation by CLN8 drives BMP biosynthesis and its loss leads to Batten disease

Loss-of-function mutations in the endoplasmic reticulum membrane protein CLN8 cause Batten disease, a neurodegenerative lysosomal storage disorder1. Together with the lysosomal enzyme CLN5, CLN8 mediates the biosynthesis of bis(monoacylglycero)phosphate (BMP), a phospholipid essential for lysosomal function and distinguished by its unique S,S stereochemistry2,3. However, the role of CLN8 in BMP synthesis has remained unclear. Here we establish that CLN8 is a glycerophosphoglycerol (GPG) acyltransferase that catalyses the stereospecific acylation of S,S-GPG to produce S,S-lysophosphatidylglycerol (LPG), the CLN5 substrate in BMP synthesis. Using cryo-electron microscopy, we resolve structures of the CLN8 homodimer in apo and substrate-bound states at 2.7 [A] resolution, revealing the active site architecture and a ping-pong acyl transfer mechanism. Batten disease-causing missense mutations impair CLN8 enzymatic activity in vitro and reduce BMP levels in a Cln8R24G knock- in mouse, whereas the Cln8mnd mouse frameshift mutation causes complete loss of BMP in vivo. Exogenous S,S-LPG, but not the R,S stereoisomer, restored BMP synthesis in CLN8- deficient cells and mice, and improved neurological phenotypes in cln8 mutant zebrafish. Together, these findings define the enzymatic function of CLN8, elucidate the biochemical basis of CLN8 Batten disease, and establish a proof-of-concept for treating it through stereospecific BMP precursor supplementation.

biochemistry↗

bioRxiv2024

In silico screening identifies SHPRH as a novel nucleosome acidic patch interactor

Nucleosomes are the fundamental unit of eukaryotic chromatin. Diverse factors interact with nucleosomes to modulate chromatin architecture and facilitate DNA repair, replication, transcription, and other cellular processes. An important platform for chromatin binding is the H2A-H2B acidic patch. Here, we used AlphaFold-Multimer to screen over 7000 human proteins for nucleosomal acidic patch binding and identify 41 potential acidic patch binders. We determined the cryo-EM structure of one hit, SHPRH, with the nucleosome at 2.8 [A]. The structure confirms the predicted acidic patch interaction, reveals that the SHPRH ATPase engages a different nucleosomal DNA location than other SF2-type ATPases, and clarifies the roles of SHPRHs domains in nucleosome recognition. Our results illustrate the use of in silico screening as a high throughput method to identify specific interaction types and expands the set of potential acidic patch binding factors. All the screening data is freely available athttps://predictomes.org/view/acidicpatch

molecular biology↗

bioRxiv2019

Enhancing autophagy by redox regulation extends lifespan in Drosophila

Dysregulation of redox homeostasis has been implicated in the ageing process and the pathology of age-related diseases. To study redox signalling by H2O2 in vivo, we established a redox-shifted model by manipulating levels of the H2O2-degrading enzyme catalase in Drosophila. Here we report that ubiquitous over-expression of catalase robustly extends lifespan in flies. As anticipated, these flies were strongly resistant to a range of oxidative stress challenges, but interestingly were sensitive to starvation, which could not be explained by differences in levels of energy reserves. This led us to explore the contribution of autophagy, which is an important mechanism for organismal survival in response to starvation. We show that autophagy is essential for the increased lifespan upon catalase up-regulation, as the survival benefits were completely abolished upon global autophagy knock-down. Furthermore, using a specific redox-inactive knock-in mutant, we highlight the in vivo role of a key regulatory cysteine residue in Atg4a, which is required for the lifespan extension in our catalase model. Altogether, these findings confirm the redox regulation of autophagy in vivo as an important modulator of longevity.

molecular biology↗

bioRxiv2018

The macrocyclizing protease butelase 1 remains auto-catalytic and reveals the structural basis for ligase activity

Plant asparaginyl endopeptidases (AEPs) are expressed as inactive zymogens that perform seed storage protein maturation upon cleavage dependent auto-activation in the low pH environment of storage vacuoles. AEPs have attracted attention for their macrocyclization reactions and have been classified as cleavage or ligation specialists. However, we have recently shown that the ability of AEPs to produce either cyclic or acyclic products can be altered by mutations to the active site region, and that several AEPs are capable of macrocyclization given favorable pH conditions. One AEP extracted from Clitoria ternatea seeds (butelase 1) is classified as a ligase rather than a protease, presenting an opportunity to test for loss of cleavage activity. Here, making recombinant butelase 1 and rescuing an Arabidopsis thaliana mutant lacking AEP, we show butelase 1 retains cleavage functions in vitro and in vivo. The in vivo rescue was incomplete, consistent with some trade-off for butelase 1 specialization toward macrocyclization. Its crystal structure showed an active site with only subtle differences from cleaving AEPs, suggesting the many differences in its peptide binding region are the source of its efficient macrocyclization. All considered, it seems either butelase 1 has not fully specialized or a requirement for auto-catalytic cleavage is an evolutionary constraint upon macrocyclizing AEPs.

biophysics↗