Biology subjects

Jones, A. M.

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

24 records · Page 2Linked to original sources

bioRxiv2020

Survival and Proliferation of T-cell Acute Lymphoblastic Leukaemia Depends on mTOR-regulated Glutamine Uptake and EAAT1 Activity

T-cell acute lymphoblastic leukaemia (T-ALL) is a cancer of the immune system. Approximately 20% of paediatric and 50% of adult T-ALL patients have refractory disease or relapse and die from the disease. To improve patient outcome new therapeutics are needed. With the aim to identify new therapeutic targets, we analysed the metabolic adaptations that T-ALL cells exhibit and found that glutamine uptake is essential for their proliferation. Isotope tracing experiments showed that glutamine fuels aspartate synthesis through the TCA cycle and that glutamine and glutamine-derived aspartate together supply three nitrogen atoms in purines and all but one atom in pyrimidine rings. We show that the glutamate-aspartate transporter EAAT1, which is normally only expressed in the CNS, is crucial for glutamine conversion to nucleotides and that T-ALL cell proliferation depends on EAAT1 function. Through this work, we identify EAAT1 as a novel therapeutic target for T-ALL treatment.

cancer biology↗

bioRxiv2019

Family permutation profiling identifies a dynamic protein domain as functionally tolerant to increased conformational entropy

To investigate whether adenylate kinase (AK) homologs differ in their functional tolerance to mutational lesions that alter dynamics, we subjected three homologs having a range of thermostabilities to random circular permutation and evaluated where new protein termini were non-disruptive to activity using a cellular selection and deep mutational scanning. Analysis of the positional tolerance to new termini, which increase local conformational entropy by breaking peptide bonds, showed that bonds were either functionally sensitive to cleavage across all three homologs, differentially sensitive, or uniformly tolerant. The mobile AMP binding domain, which displays the highest calculated contact energies (frustration), presented the greatest tolerance to new termini across all AKs. In contrast, retention of function in the lid and core domains was more dependent upon AK melting temperature. Thus, regions of high energetic frustration tolerated increases in conformational entropy in a manner that was less dependent on thermostability than regions of lower frustration. Our results suggest that family permutation profiling identifies primary structure that has been selected by evolution for high frustration that is critical to enzymatic activity. They also illustrate how deep mutational scanning can be applied to protein homologs in parallel to learn how topology and function govern mutational tolerance.

biochemistry↗

bioRxiv2019

A decoy heterotrimeric Gα protein has substantially reduced nucleotide binding but retains nucleotide-independent interactions with its cognate RGS protein and Gβγ dimer

Plants uniquely have a family of proteins called extra-large G proteins (XLG) that share homology in their C-terminal half with the canonical G subunits; we carefully detail here that Arabidopsis XLG2 lacks critical residues requisite for nucleotide binding and hydrolysis which is consistent with our quantitative analyses. Based on microscale thermophoresis, Arabidopsis XLG2 binds GTP{gamma}S with an affinity 100-1000 times lower than that to canonical G subunits. This means that given the concentration range of guanine nucleotide in plant cells, XLG2 is not likely bound by GTP in vivo. Homology modeling and molecular dynamics simulations provide a plausible mechanism for the poor nucleotide binding affinity of XLG2. Simulations indicate substantially stronger salt bridge networks formed by several key amino-acid residues of AtGPA1 which are either misplaced or missing in XLG2. These residues in AtGPA1 not only maintain the overall shape and integrity of the apoprotein cavity but also increase the frequency of favorable nucleotide-protein interactions in the nucleotide-bound state. Despite this loss of nucleotide dependency, XLG2 binds the RGS domain of AtRGS1 with an affinity similar to the Arabidopsis AtGPA1 in its apo-state and about 2 times lower than AtGPA1 in its transition state. In addition, XLG2 binds the G{beta}{gamma} dimer with an affinity similar to that of AtGPA1. XLG2 likely acts as a dominant negative G protein to block G protein signaling. We propose that XLG2, independent of guanine nucleotide binding, regulates the active state of the canonical G protein pathway directly by sequestering G{beta}{gamma} and indirectly by promoting heterodimer formation.

biochemistry↗

bioRxiv2019

Receptor-Like Kinase Phosphorylation of Arabidopsis Heterotrimeric G-Protein Gα - Subunit AtGPA1

As molecular on-off switches, heterotrimeric G protein complexes, comprised of a G subunit and an obligate G{beta}{gamma} dimer, transmit extracellular signals received by G protein- coupled receptors (GPCRs) to cytoplasmic targets that respond to biotic and abiotic stimuli. Signal transduction is modulated by phosphorylation of GPCRs and G protein complexes. In Arabidopsis thaliana, the G subunit AtGPA1 is phosphorylated by the receptor-like kinase (RLK) BRI1-ASSOCIATED Kinase 1 (BAK1), but the extent that other RLKs phosphorylates AtGPA1 is unknown. We mapped 22 trans-phosphorylation sites on AtGPA1 by 12 RLKs hypothesized to act in the Arabidopsis G protein signaling pathway. Cis-phosphorylation sites on these RLKs were also identified. BRI1, BAK1, and SERK1 have been reported as Ser/Thr and Tyr dual specificity kinases. We identified 4 more dual specificity kinases: IOS1, PSY1R, PEPR1, and AT2G37050. Multiple sites are present in the core AtGPA1 functional units, including pSer52 and pThr53 of the conserved P-loop that directly binds nucleotide/phosphate, pThr164 and pSer175 from E helix in the intramolecular domain interface for nucleotide exchange and GTP hydrolysis, and pThr193 or pThr194 in Switch I (SwI) that coordinates nucleotide exchange and protein partner binding. Several AtGPA1 S/T phosphorylation sites are nucleotide-dependent phosphorylation patterns, such as S52/T53 in the P-loop and T193 and/or T194 in SwI.

plant biology↗

bioRxiv2019

Urbilaterian origin and evolution of sNPF-type neuropeptide signalling

Physiology and behaviour are controlled by neuropeptide signalling systems comprising peptide ligands and cognate receptors. Molecular phylogenetics combined with experimental identification of neuropeptide-receptor pairs has revealed that many neuropeptide signalling systems originated in the urbilaterian common ancestor of protostomes and deuterostomes. Neuropeptide-Y/neuropeptide-F (NPY/NPF)-type signalling is one such example, whereas NPY/NPF-related short-NPF (sNPF)-type signalling has hitherto only been identified in protostomes. Here we report the discovery of a neuropeptide (pQDRSKAMQAERTGQLRRLNPRF-NH2) that is the ligand for an sNPF-type receptor in a deuterostome, the starfish Asterias rubens (Phylum Echinodermata). Informed by phylogenetic analysis of sequence data, we conclude that the paralogous NPY/NPF-type and sNPF-type signalling systems originated in Urbilateria but NPY/NPF-type signalling was lost in echinoderms. Furthermore, we present evidence that sNPF-type peptides are orthologs of vertebrate prolactin-releasing peptides. Our findings demonstrate the importance of experimental studies on echinoderms for reconstructing the evolutionary history of neuropeptide signalling systems.

neuroscience↗

bioRxiv2019

Expanding the Zebrafish Genetic Code through Site-Specific Introduction of Diazirine-lysine, Bicyclononyne-lysine and Azido-lysine

Site-specific incorporation of un-natural amino acids (UNAA) is a powerful approach to engineer and understand protein function [1-4]. Site-specific incorporation of UNAAs is achieved through repurposing the amber codon (UAG) as a sense codon for the UNAA, a tRNACUA that base pairs with an UAG codon in the mRNA and an orthogonal amino-acyl tRNA synthetase (aaRS) that charges the tRNACUA with the UNAA [5, 6]. Here, we report expansion of the zebrafish genetic code to incorporate the UNAAs, Azido-lysine (AzK), bicyclononyne-lysine (BCNK), and Diazirine-lysine (AbK) into green fluorescent protein (GFP) and Glutathione-S-transferase (GST). We also present proteomic evidence for UNAA incorporation into GFP. Our work sets the stage for the use of UNAA mutagenesis to investigate and engineer protein function in zebrafish.

synthetic biology↗