A quantitative screen for metabolic enzyme structures reveals patterns of assembly across the yeast metabolic network.

Despite the proliferation of proteins that can form filaments or phase-separated condensates, it remains unclear how this behavior is distributed over biological networks. We have found that 60 of the 440 yeast metabolic enzymes robustly form structures, including 10 that assemble within mitochondria. Additionally, the ability to assemble is enriched at branch points on several metabolic pathways. The assembly of enzymes at the first branch point in de novo purine biosynthesis is coordinated, hierarchical, and based on their position within the pathway, while the enzymes at the second branch point are recruited to RNA stress granules. Consistent with distinct classes of structures being deployed at different control points in a pathway, we find that the first enzyme in the pathway, PRPP synthetase, forms evolutionarily conserved filaments that are sequestered in the nucleus in higher eukaryotes. These findings provide a roadmap for identifying additional conserved features of metabolic regulation by condensates/filaments

A quantitative screen for metabolic enzyme structures reveals patterns of assembly across the yeast metabolic network

© The Author(s), 2019. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Noree, C., Begovich, K., Samilo, D., Broyer, R., Monfort, E., & Wilhelm, J. E. A quantitative screen for metabolic enzyme structures reveals patterns of assembly across the yeast metabolic network. Molecular Biology of the Cell, 30(21), (2019): 2721-2736, doi:10.1091/mbc.E19-04-0224.Despite the proliferation of proteins that can form filaments or phase-separated condensates, it remains unclear how this behavior is distributed over biological networks. We have found that 60 of the 440 yeast metabolic enzymes robustly form structures, including 10 that assemble within mitochondria. Additionally, the ability to assemble is enriched at branch points on several metabolic pathways. The assembly of enzymes at the first branch point in de novo purine biosynthesis is coordinated, hierarchical, and based on their position within the pathway, while the enzymes at the second branch point are recruited to RNA stress granules. Consistent with distinct classes of structures being deployed at different control points in a pathway, we find that the first enzyme in the pathway, PRPP synthetase, forms evolutionarily conserved filaments that are sequestered in the nucleus in higher eukaryotes. These findings provide a roadmap for identifying additional conserved features of metabolic regulation by condensates/filaments.We thank Douglass Forbes for comments on the manuscript, Susanne Rafelski for the gift of the pVTU-mito-dsRed plasmid, and Brian Zid for the gift of the pKT-mNeonGreen plasmid. Work at the Wilhelm lab was supported by a grant from the Hughes Collaborative Innovation Award program of the Howard Hughes Medical Institute and the James Wilhelm Memorial Fund. Kyle Begovich is a Howard Hughes Medical Institute Gilliam Fellow

Identification of novel filament-forming proteins in Saccharomyces cerevisiae and Drosophila melanogaster

A screen for GFP-tagged yeast proteins that can assemble into visible structures reveals four new filamentous structures in the cytoplasm formed by metabolic enzymes and translation factors

Spatial organization of mRNA regulation and metabolic activity

Organization of biological processes is a central principle of cell biology. However, until recently, the context of this organization has largely centered on membrane-bound organelles and their internal biochemistry. Recent discoveries of intracellular structures that organize biochemical processes such as P bodies, purinosomes, and self-assembling metabolic enzymes suggests there is much to be uncovered in the studies of cytoplasmic organization. This thesis focuses on two mechanisms for organizing the cytoplasm: one involving the spatial regulation of key mRNA processing events in early development and the other involving polymerization of metabolic enzymes and the role it plays in connecting metabolic regulation to broader areas of cell biology. The spatial regulation of mRNA processing events is largely dependent on the role of the mRNA-associated ribonucleoprotein (RNP) complex that functions to regulate transcript translation and stability. We focus on one such RNP, Cup, that has been previously described as a translational repressor for oskar mRNA, the transcript that is critical for anterior-posterior patterning in Drosophila development. Here, we identify that Cup is also required for oskar mRNA stability. Conversely, we will also show a novel pathway for mRNA degradation. Previous studies have identified the role of the Pan gu kinase complex in activating the translation of the mRNA degradation machinery at the maternal-to-zygotic transition, where maternally loaded transcripts are degraded as a precursor to zygotic transcriptional control. We identify a parallel pathway that acts in concert to destabilize these maternal transcripts through ubiquitin-mediated degradation of the associated RNPs. Switching to a different mechanism of cytoplasmic organization, we will reveal the self-assembling property of the metabolic enzyme PRPP synthetase (PRPS), the enzyme responsible for synthesizing the substrate for nucleotide biosynthesis. We will also show the defects in cellular actin organization associated with the mutations in PRPS leading to a disease-state. Furthermore, we will identify that the inhibitor of PRPS associates with the polymerized form of PRPS and also with the actin cytoskeleton; this suggests a novel method of regulation and possible mechanism behind PRPS diseases

Spatial organization of mRNA regulation and metabolic activity

Organization of biological processes is a central principle of cell biology. However, until recently, the context of this organization has largely centered on membrane-bound organelles and their internal biochemistry. Recent discoveries of intracellular structures that organize biochemical processes such as P bodies, purinosomes, and self-assembling metabolic enzymes suggests there is much to be uncovered in the studies of cytoplasmic organization. This thesis focuses on two mechanisms for organizing the cytoplasm: one involving the spatial regulation of key mRNA processing events in early development and the other involving polymerization of metabolic enzymes and the role it plays in connecting metabolic regulation to broader areas of cell biology. The spatial regulation of mRNA processing events is largely dependent on the role of the mRNA-associated ribonucleoprotein (RNP) complex that functions to regulate transcript translation and stability. We focus on one such RNP, Cup, that has been previously described as a translational repressor for oskar mRNA, the transcript that is critical for anterior-posterior patterning in Drosophila development. Here, we identify that Cup is also required for oskar mRNA stability. Conversely, we will also show a novel pathway for mRNA degradation. Previous studies have identified the role of the Pan gu kinase complex in activating the translation of the mRNA degradation machinery at the maternal-to-zygotic transition, where maternally loaded transcripts are degraded as a precursor to zygotic transcriptional control. We identify a parallel pathway that acts in concert to destabilize these maternal transcripts through ubiquitin-mediated degradation of the associated RNPs. Switching to a different mechanism of cytoplasmic organization, we will reveal the self-assembling property of the metabolic enzyme PRPP synthetase (PRPS), the enzyme responsible for synthesizing the substrate for nucleotide biosynthesis. We will also show the defects in cellular actin organization associated with the mutations in PRPS leading to a disease-state. Furthermore, we will identify that the inhibitor of PRPS associates with the polymerized form of PRPS and also with the actin cytoskeleton; this suggests a novel method of regulation and possible mechanism behind PRPS diseases

Identification of novel filament-forming proteins in Saccharomyces cerevisiae and Drosophila melanogaster.

The discovery of large supramolecular complexes such as the purinosome suggests that subcellular organization is central to enzyme regulation. A screen of the yeast GFP strain collection to identify proteins that assemble into visible structures identified four novel filament systems comprised of glutamate synthase, guanosine diphosphate-mannose pyrophosphorylase, cytidine triphosphate (CTP) synthase, or subunits of the eIF2/2B translation factor complex. Recruitment of CTP synthase to filaments and foci can be modulated by mutations and regulatory ligands that alter enzyme activity, arguing that the assembly of these structures is related to control of CTP synthase activity. CTP synthase filaments are evolutionarily conserved and are restricted to axons in neurons. This spatial regulation suggests that these filaments have additional functions separate from the regulation of enzyme activity. The identification of four novel filaments greatly expands the number of known intracellular filament networks and has broad implications for our understanding of how cells organize biochemical activities in the cytoplasm

Human retinal ganglion cell neurons generated by synchronous BMP inhibition and transcription factor mediated reprogramming.

In optic neuropathies, including glaucoma, retinal ganglion cells (RGCs) die. Cell transplantation and endogenous regeneration offer strategies for retinal repair, however, developmental programs required for this to succeed are incompletely understood. To address this, we explored cellular reprogramming with transcription factor (TF) regulators of RGC development which were integrated into human pluripotent stem cells (PSCs) as inducible gene cassettes. When the pioneer factor NEUROG2 was combined with RGC-expressed TFs (ATOH7, ISL1, and POU4F2) some conversion was observed and when pre-patterned by BMP inhibition, RGC-like induced neurons (RGC-iNs) were generated with high efficiency in just under a week. These exhibited transcriptional profiles that were reminiscent of RGCs and exhibited electrophysiological properties, including AMPA-mediated synaptic transmission. Additionally, we demonstrated that small molecule inhibitors of DLK/LZK and GCK-IV can block neuronal death in two pharmacological axon injury models. Combining developmental patterning with RGC-specific TFs thus provided valuable insight into strategies for cell replacement and neuroprotection

Human retinal ganglion cell neurons generated by synchronous BMP inhibition and transcription factor mediated reprogramming

Abstract In optic neuropathies, including glaucoma, retinal ganglion cells (RGCs) die. Cell transplantation and endogenous regeneration offer strategies for retinal repair, however, developmental programs required for this to succeed are incompletely understood. To address this, we explored cellular reprogramming with transcription factor (TF) regulators of RGC development which were integrated into human pluripotent stem cells (PSCs) as inducible gene cassettes. When the pioneer factor NEUROG2 was combined with RGC-expressed TFs (ATOH7, ISL1, and POU4F2) some conversion was observed and when pre-patterned by BMP inhibition, RGC-like induced neurons (RGC-iNs) were generated with high efficiency in just under a week. These exhibited transcriptional profiles that were reminiscent of RGCs and exhibited electrophysiological properties, including AMPA-mediated synaptic transmission. Additionally, we demonstrated that small molecule inhibitors of DLK/LZK and GCK-IV can block neuronal death in two pharmacological axon injury models. Combining developmental patterning with RGC-specific TFs thus provided valuable insight into strategies for cell replacement and neuroprotection

Inhibition of GCK-IV kinases dissociates cell death and axon regeneration in CNS neurons.

Axon injury is a hallmark of many neurodegenerative diseases, often resulting in neuronal cell death and functional impairment. Dual leucine zipper kinase (DLK) has emerged as a key mediator of this process. However, while DLK inhibition is robustly protective in a wide range of neurodegenerative disease models, it also inhibits axonal regeneration. Indeed, there are no genetic perturbations that are known to both improve long-term survival and promote regeneration. To identify such a neuroprotective target, we conducted a set of complementary high-throughput screens using a protein kinase inhibitor library in human stem cell-derived retinal ganglion cells (hRGCs). Overlapping compounds that promoted both neuroprotection and neurite outgrowth were bioinformatically deconvoluted to identify specific kinases that regulated neuronal death and axon regeneration. This work identified the role of germinal cell kinase four (GCK-IV) kinases in cell death and additionally revealed their unexpected activity in suppressing axon regeneration. Using an adeno-associated virus (AAV) approach, coupled with genome editing, we validated that GCK-IV kinase knockout improves neuronal survival, comparable to that of DLK knockout, while simultaneously promoting axon regeneration. Finally, we also found that GCK-IV kinase inhibition also prevented the attrition of RGCs in developing retinal organoid cultures without compromising axon outgrowth, addressing a major issue in the field of stem cell-derived retinas. Together, these results demonstrate a role for the GCK-IV kinases in dissociating the cell death and axonal outgrowth in neurons and their druggability provides for therapeutic options for neurodegenerative diseases