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Herzog: Biological Overview | References

Prion-like proteins can assume distinct conformational and physical states in the same cell. Sequence analysis suggests that prion-like proteins are prevalent in various species; however, it remains unclear what functional space they occupy in multicellular organisms. This study reports the identification of a prion-like protein, Herzog (CG5830), through a multimodal screen in Drosophila melanogaster. Herzog functions as a membrane-associated phosphatase and controls embryonic patterning, likely being involved in TGF-beta/BMP and FGF/EGF signaling pathways. Remarkably, monomeric Herzog, a protein-serine/threonine phosphatase, is enzymatically inactive and becomes active upon amyloid-like assembly. The prion-like domain of Herzog is necessary for both its assembly and membrane targeting. Removal of the prion-like domain impairs activity, while restoring assembly on the membrane using a heterologous prion-like domain and membrane-targeting motif can restore phosphatase activity. This study provides an example of a prion-like domain that allows an enzyme to gain essential functionality via amyloid-like assembly to control animal development (Nil, 2019).

Most proteins adopt one specific conformation in a cell dictated by their primary amino acid sequence. The relationship between a protein's sequence and one structure has in recent years been complemented by an exciting alternative. One group of proteins, called prion-like proteins, can assume distinct conformational states in the same cell, and often one of these states leads to a higher-order state that can vary in physical nature—from liquid to hydrogel to non-amyloid or amyloid-like aggregates (Wu, 2016). Prion-like proteins often contain a modular domain, called a prion-like domain (PrD), which is necessary and sufficient for higher-order state (Wickner, 2000). Prion-like proteins are important regulators of various physiological processes in diverse species (Jakobson, 2018), such as adaptation to changing environmental conditions, immune response, and memory formation. Recent in silico analyses suggest that proteins with prion-like domains are prevalent in higher eukaryotic proteomes and may occupy broader functional space. However, with few exceptions, in most cases it remains unclear what the functional relevance is, if any, of the prion-like domains (Nil, 2019).

The establishment of cell fates during tissue specification and embryonic patterning are achieved by regulated activation and deactivation of various signaling pathways. Protein phosphorylation, a common dynamic post-translational modification (PTM), lies at the heart of developmental signaling pathways. Cells precisely control the state and the amplitude of signaling pathways by adding, stabilizing, or removing phosphate groups through the opposing activity of protein kinases and phosphatases. The complexity and specificity of phosphorylation is generally associated with the diversity of the kinases, and almost 2% of the eukaryotic protein coding genes encode for protein kinases. In contrast to kinases, a small number, ∼0.6% of genes, encode for protein phosphatases, and often in vitro multiple phosphatases act on the same substrate or the same phosphatase can act on multiple substrates, implying a low level of substrate specificity and regulation. Despite this apparent promiscuity and redundancy, loss of phosphatases often leads to very specific phenotypes. Therefore, unlike kinases, it remains unclear how seemingly promiscuous phosphatases achieve substrate specificity or control activity both in time and space (Nil, 2019).

A small-scale systematic screen of the Drosophila melanogaster proteome led to the identification of five potential prion-like proteins . One of these candidates, which was named Herzog (Hzg), is a functionally uncharacterized gene (CG5830), with homology to the haloacid dehalogenase (HAD) subfamily small CTD phosphatases (SCPs). Hzg, a membrane-associated phosphatase, is a maternal effect gene required for proper establishment of segment polarity in the embryos. Hzg is expressed ubiquitously throughout embryonic development but changes from monomers to aggregates with features of amyloids during gastrulation, and this state change leads to a gain of phosphatase activity. The phosphatase activity requires both membrane localization and amyloid-like fibrillization through its N-terminal prion-like domain. Taken together, this study provides a definitive example of how a prion-like domain can control embryonic development by orchestrating phosphatase activity in space and time (Nil, 2019).

In silico sequence-based analysis indicates that proteins with prion-like domains are quite abundant in all branches of life. However, with a few exceptions, the functional relevance of prion-like domains in multicellular eukaryotes remains largely unknown. This stduy reports that Hzg, through its N-terminal prion-like domain, changes from an inactive monomer to an active amyloid-like aggregate on the membrane during gastrulation, likely to control segment polarity. Although detailed structural information is lacking, the fact that substrate recognition does not require the N-terminal domain, but phosphatase activity does, and that the only fibrillar (recombinant) protein has enzymatic activity suggest that the conformational change into an amyloid like aggregate allows Hzg to be catalytically active (Nil, 2019).

Hzg belongs to HAD family of phosphatases, an ancient large class of enzymes, with unknown functions. For most phosphatases, a recognizable regulatory domain apart from their catalytic domains controls enzymatic function by controlling compartmentalization or substrate binding. HAD subfamily of SCPs (to which Hzg belongs), like prokaryotic HADs, contains a single catalytic domain without any of the recognizable structural regulatory domains. They also do not have a cap module in their active site, which is used by other HAD family members for substrate selectivity. Therefore, Hzg likely utilizes the conformational reorganization associated with amyloid-like fold to control phosphatase activity. The use of a prion-like domain to induce a conformational change and thereby catalytic activity adds to the repertoire of regulatory modes of enzymatic activity. Considering that several phosphatases and kinases in Drosophila and in other species harbor prion-like domains, it is possible that the aggregation-based modulation of enzymatic activity may not be rare. Intriguingly, the prion-like domain domain of Hzg is not only important for aggregation but also for its membrane localization. It is uncertain how Hzg prion-like domain targets the protein to the membrane and whether aggregation occurs in the membrane or whether preformed aggregates are recruited to the membrane. Whatever the mechanism, membrane localization and regulated aggregation and dissolution can confine the phosphatase activity in space and time, allowing a phosphatase to act in a substrate-specific manner. A key question for the future is how Hzg aggregation is regulated (Nil, 2019).

The screen was biased toward identifying prion-like proteins that form stable assemblies. Nonetheless, it begs the question as to why an enzyme forms an amyloid-like state, seemingly a stable state, to control dynamic signaling pathways. One possibility is that unlike liquid, gel, or glass-like assemblies, where the proteins are organized randomly, in an amyloid-like state the ordered assembly and as a result the ordered organization of the catalytic domain can offer a more reliable interaction between enzyme and substrate. Mechanistically, it is possible that the catalytic domain is hidden in the monomer and that the amyloid-like assembly reveals that catalytic domain. It is hypothesized that since the substrate binding and catalytic domain (M domain) is outside the N-terminal prion-like domain, the substrate does not need to access the core of the fiber. Conformational changes associated with amyloid-like fibrilization simply orient the substrate binding and catalytic domain to the surface of the fiber. Alternatively, three-dimensional (3D) domain swapping associated with amyloid-like assembly may turn inactive monomers to an enzymatically active oligomer or polymer. Indeed, almost a decade ago, Eisenberg and colleagues elegantly showed that two inactive monomers of bovine ribonuclease A can be arranged into an enzymatically active amyloid-like aggregate (Sambashivan, 2005). It is speculated that Hzg represents a natural example of turning an inactive enzyme active via amyloid-like assembly (Nil, 2019).

In addition to conformational alteration, what are the consequences of adopting an amyloid-like state with respect to function? Historically, amyloids are thought to be irreversible primarily based on in vitro studies of disease-causing amyloids that originate from misfolded or truncated proteins. However, over the years it has emerged that cellular proteins such as enzymes, hormone peptides, mRNA, and DNA-binding proteins can adopt amyloid-like states and disassemble under normal cellular conditions. Moreover, from recent atomic-level structural analyses of various amyloid-like proteins, it is also emerging that in addition to canonical cross-β sheet, there are also α-helical amyloid-like states and amyloid-like states with charged residues in the core that confer flexibility. Although, the physiological relevance of such diversity and structural flexibility remains unclear, it is speculated that flexibility may allow an enzyme like Hzg to form a stable assembly that nonetheless can be taken apart. Development, which needs to accommodate the changing environment, might utilize such molecular stability and flexibility to tune the time course of development. With respect to heterogeneity in structure, as stated earlier, the number of phosphatases encoded by the genome is relatively small compared to kinases considering that almost one-third of all proteins in the cell are regulated by phosphorylation. The structural flexibility may help with expanding the functional space (Nil, 2019).

Proteomics analysis followed by pairwise interaction assays for Hzg-interacting partners suggest that there could be at least seven putative substrates for Hzg; Babo, Dah, Irk, Pch2, Ras64B, Sax, and Src64B. Multiple substrates implies that the phenotypes of hzg mutants may be pleotropic. Considering the multiple putative membrane-associated substrates, biochemical function, and mobility of Hzg aggregates on the membrane, he following speculative model is proposed: Hzg forms functional amyloidogenic aggregates on the membrane during gastrulation, and these aggregates moves along the membrane, allowing Hzg to act on different targets over time (Nil, 2019).

Mutant embryos expressing only the N-terminal prion-like domain of Hzg show defects in segment polarity, and this phenotype is reminiscent of mutations of wingless (wg) signaling antagonists such as zw3/GSK, D-axin, and D-Apc2. However, in the current proteomics analysis, no interaction of Hzg was seen with canonical Wg-signaling components, suggesting that Hzg may influence wg signaling indirectly. It is possible that the action of Hzg in TGF-β/BMP and EGF/FGF signaling (Ras64B and Src64B) pathways that are known to cross talk with the wg-signaling pathway contributes to the mutant phenotype. Further work would be necessary to obtain detailed mechanistic insight into the role of Hzg in embryonic development (Nil, 2019).

Search PubMed for articles about Drosophila Hertzog

Jakobson, C. M. and Jarosz, D. F. (2018). Organizing biochemistry in space and time using prion-like self-assembly. Curr Opin Syst Biol 8: 16-24. PubMed ID: 29725624

Nil, Z., Millan, R. H., Gerbich, T., Leal, P., Yu, Z., Saraf, A., Sardiu, M., Lange, J. J., Yi, K., Unruh, J., Slaughter, B. and Si, K. (2019). Amyloid-like assembly activates a phosphatase in the developing Drosophila embryo. Cell 178(6): 1403-1420. PubMed ID: 31491385

Sambashivan, S., Liu, Y., Sawaya, M. R., Gingery, M. and Eisenberg, D. (2005). Amyloid-like fibrils of ribonuclease A with three-dimensional domain-swapped and native-like structure. Nature 437(7056): 266-269. PubMed ID: 16148936

Wickner, R. B., Taylor, K. L., Edskes, H. K. and Maddelein, M. L. (2000). Prions: Portable prion domains. Curr Biol 10(9): R335-337. PubMed ID: 10801430

Wu, H. and Fuxreiter, M. (2016). The structure and dynamics of higher-order assemblies: Amyloids, signalosomes, and granules. Cell 165(5): 1055-1066. PubMed ID: 27203110

date revised: 15 December 2019

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