Biology subjects

Andrews, A.

Publications and source records attributed to Andrews, A..

3 recordsLinked to original sources

bioRxiv2018

Two factor authorization: Asf1 mediates crosstalk between H3 K14 and K56 acetylation

Acetylation of histones plays a critical role in maintaining the epigenetic state of the eukaryotic cell. One such acetylation site critical for DNA damage repair is H3K56ac. In Saccharomyces cerevisiae, H3K56ac is thought to be driven mainly by Rtt109, a lysine acetyltransferase (KAT) that associates with the histone chaperones Vps75 and Asf1. Both of these chaperones can increase the specificity of histone acetylation by Rtt109, but neither alter the selectivity. It has been shown that histones extracted from cells (Drosophila), presumably containing pre-acetylated histones, can incorporate higher amounts of H3K56ac relative to recombinant non-acetylated histones. We hypothesized that histone pre-acetylation and histone chaperones could function together to drive acetylation of H3K56. In the present study, we test this hypothesis using a series of singly acetylated histones to determine the impact of crosstalk on enzyme selectivity. Our data suggest that crosstalk between acetylation sites plays a major role in altering the selectivity of Rtt109-Vps75 and that the histone chaperone Asf1 mediates this crosstalk. Specifically, we show that H3K14ac/H4 functions with Asf1 to drive H3K56ac by Rtt109-Vps75. We identified an acidic patch in Asf1 that mediates this cross-talk and show that mutations to this region can alter the Asf1 mediated crosstalk that changes Rtt109-Vps75 selectivity. These data explain the genetic link between Gcn5, which acetylates H3K14 and Rtt109. More broadly these data demonstrate that acetylation sites can dictate site selectivity even in the absence of a bromodomain and helps to explain the limited complexity that has been observed of the histone post-translational modifications patterns by global proteomic studies.

biochemistry↗

bioRxiv2018

T Cell Activation Triggers Reversible Inosine-5'-Monophosphate Dehydrogenase Assembly

T cell-mediated adaptive immunity requires naive, unstimulated T cells to transition from a quiescent metabolic state into a highly proliferative state upon T cell receptor engagement. This complex process depends on transcriptional changes mediated by Ca2+-dependent NFAT signaling, mTOR-mediated signaling and increased activity of the guanine nucleotide biosynthetic enzyme inosine-5-monophosphate (IMP) dehydrogenase (IMPDH). Inhibitors of these pathways serve as potent immunosuppressants. Unexpectedly, we discovered that all three pathways converge to promote the assembly of IMPDH protein into micron-scale macromolecular filamentous structures in response to T cell activation. Assembly is post-transcriptionally controlled by mTOR and the Ca2+ influx regulator STIM1. Furthermore, IMPDH assembly and catalytic activity were negatively regulated by guanine nucleotide levels, suggesting a negative feedback loop that limits biosynthesis of guanine nucleotides. Filamentous IMPDH may be more resistant to this inhibition, facilitating accumulation of the higher GTP levels required for T cell proliferation.

immunology↗

bioRxiv2017

IMPDH polymers accommodate both catalytically active and inactive conformations

Several metabolic enzymes undergo reversible polymerization into macromolecular assemblies. The function of these assemblies is often unclear but in some cases they regulate enzyme activity and metabolic homeostasis. The guanine nucleotide biosynthetic enzyme inosine monophosphate dehydrogenase (IMPDH) forms octamers that polymerize into helical chains. In mammalian cells, IMPDH filaments can associate into micron-length assemblies. Polymerization and enzyme activity are regulated in part by binding of purine nucleotides to an allosteric regulatory domain. ATP promotes octamer polymerization, whereas GTP promotes a compact, inactive conformation whose ability to polymerize is unknown. An open question is whether polymerization directly alters IMPDH catalytic activity. To address this, we identified point mutants of human IMPDH2 that either prevent or promote polymerization. Unexpectedly, we found that polymerized and non-assembled forms of IMPDH have comparable catalytic activity, substrate affinity, and GTP sensitivity and validated this finding in cells. Electron microscopy revealed that substrates and allosteric nucleotides shift the equilibrium between active and inactive conformations in both the octamer and the filament. Unlike other metabolic filaments, which selectively stabilize active or inactive conformations, IMPDH filaments accommodate multiple states. Thus, although polymerization alone does not impact catalytic activity, substrate availability and purine balance dramatically affect IMPDH filament architecture.

biochemistry↗