Biology subjects

Irwin, N. A. T.

Publications and source records attributed to Irwin, N. A. T..

3 recordsLinked to original sources

bioRxiv2024

PHLPP2 is a pseudophosphatase that lost activity in the metazoan ancestor

The phosphoinositide 3-kinase (PI3K) pathway is a major regulator of cell and organismal growth. Consequently, hyperactivation of PI3K and its downstream effector kinase, Akt, is observed in many human cancers. PH domain leucine-rich repeat-containing protein phosphatases (PHLPP), two paralogous members of the metal-dependent protein phosphatase family, have been reported as negative regulators of Akt signaling and, therefore, tumor suppressors. However, the stoichiometry and identity of the bound metal ion(s), mechanism of action, and enzymatic specificity of these proteins are not known. Seeking to fill these gaps in our understanding of PHLPP biology, we unexpectedly discovered that PHLPP2 has no catalytic activity against the regulatory phosphorylation sites of Akt, nor the generic substrate para -nitrophenylphosphate. Instead, we found that PHLPP2 is a pseudophosphatase with a single zinc ion bound in its catalytic center. Furthermore, we found that current cancer genomics data do not support the proposed role of PHLPP1 or PHLPP2 as tumor suppressors. Phylogenetic analyses revealed an ancestral phosphatase that arose more than 1 Mya, but that lost activity at the base of the metazoan lineage. In summary, our results provide a molecular explanation for the inconclusive results that have hampered research on PHLPP and argue for a new focus on non-catalytic roles of PHLPP1 and PHLPP2. Significance StatementPHLPP1 and PHLPP2 have previously been reported as protein phosphatases that specifically inactivate Akt, a pro-growth and survival kinase hyperactivated in many human cancers. Unexpectedly, we found that purified PHLPP2 has no detectable enzymatic activity in vitro, an observation which can be rationalized by its unusual active site, which has diverged significantly from that of canonical metal-dependent phosphatases. Furthermore, we show that cancer genomics do not support a role for either PHLPP1 or PHLPP2 in cancer. Our findings argue for the exploration of alternative hypotheses regarding the role of PHLPP in Akt signaling and cancer, with a focus on its non-catalytic functions.

biochemistry↗

bioRxiv2023

Self-assembling viral histones unravel early nucleosome evolution

Nucleosomes are a core-component of eukaryotic nuclei, forming the structural basis of chromatin and co-ordinating processes from gene expression to chromosome segregation. Composed of a DNA-protein complex consisting of the four individual histones, H2A, H2B, H3, and H4, the nucleosome and its associated functions were key innovations during eukaryotic evolution1,2. However, functional constraints and the extinction of stem-eukaryotes have concealed how these dynamic systems evolved from simpler histone homologues in Archaea3-5. Viral histones have also previously been identified and are thought to reflect an ancestral state as they often comprise multiple histone paralogues arranged within a single protein, termed histone repeats6-11. Here, using viruses as an alternative source of variation, we expand the known diversity of histones and develop an empirical hypothesis for the origin of the nucleosome. Our analysis identified hundreds of histones with variable domain repeat configurations including histone singlets, doublets, triplets, and quadruplets, the latter comprising the four core histones arranged in series. Viral histone repeats consistently branch between Archaea and eukaryotes in phylogenetic trees and display intermediate functions, self-assembling into eukaryotic-like nucleosomes that stack into archaeal-like oligomers capable of impacting genomic activity and condensing DNA. The linkers conjoining the histone repeats also facilitate nucleosome formation and can promote the assembly of eukaryotic nucleosomes in the bacterium, Escherichia coli. Combining these data, we hypothesize that viral histone repeats represent molecular relics acquired by viruses from stem-eukaryotes during eukaryogenesis and suggest that nucleosome evolution may have proceeded through histone repeat intermediates.

evolutionary biology↗

bioRxiv2022

Shuffled ATG8 interacting motifs form an ancestral bridge between UFMylation and C53-mediated autophagy

UFMylation mediates the covalent modification of substrate proteins with UFM1 (Ubiquitin-fold modifier 1) and regulates the selective degradation of endoplasmic reticulum (ER) via autophagy (ER-phagy) to maintain ER homeostasis. Specifically, collisions of the ER-bound ribosomes trigger ribosome UFMylation, which in turn activates C53-mediated autophagy that clears the toxic incomplete polypeptides. C53 has evolved non-canonical shuffled ATG8 interacting motifs (sAIMs) that are essential for ATG8 interaction and autophagy initiation. Why these non-canonical motifs were selected during evolution, instead of canonical ATG8 interacting motifs remains unknown. Here, using a phylogenomics approach, we show that UFMylation is conserved across the eukaryotes and secondarily lost in fungi and some other species. Further biochemical assays have confirmed those results and showed that the unicellular algae, Chlamydomonas reinhardtii has a functional UFMylation machinery, overturning the assumption that this process is linked to multicellularity. Our conservation analysis also revealed that UFM1 co-evolves with the sAIMs in C53, reflecting a functional link between UFM1 and the sAIMs. Using biochemical and structural approaches, we confirmed the interaction of UFM1 with the C53 sAIMs and found that UFM1 and ATG8 bound to the sAIMs in a different mode. Conversion of sAIMs into canonical AIMs prevented binding of UFM1 to C53, while strengthening ATG8 interaction. This led to the autoactivation of the C53 pathway and sensitized Arabidopsis thaliana to ER stress. Altogether, our findings reveal an ancestral toggle switch embodied in the sAIMs that regulates C53-mediated autophagy to maintain ER homeostasis.

plant biology↗