Cdc14 protein phosphatases play an important role in plant infection by several fungal pathogens. This and other properties of Cdc14 enzymes make them an intriguing target for development of new antifungal crop treatments. Active site architecture and substrate specificity of Cdc14 from the model fungus
Protein arginylation mediated by arginyltransferase ATE1 is a key regulatory process essential for mammalian embryogenesis, cell migration, and protein regulation. Despite decades of studies, very little is known about the specificity of ATE1-mediated target site recognition. Here, we used
- PAR ID:
- 10153318
- Publisher / Repository:
- Nature Publishing Group
- Date Published:
- Journal Name:
- Scientific Reports
- Volume:
- 8
- Issue:
- 1
- ISSN:
- 2045-2322
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
Abstract Saccharomyces cerevisiae (ScCdc14) are well-defined and unique among characterized phosphatases. Cdc14 appears absent from some model plants. However, the extent of conservation of Cdc14 sequence, structure, and specificity in fungal plant pathogens is unknown. We addressed this by performing a comprehensive phylogenetic analysis of the Cdc14 family and comparing the conservation of active site structure and specificity among a sampling of plant pathogen Cdc14 homologs. We show that Cdc14 was lost in the common ancestor of angiosperm plants but is ubiquitous in ascomycete and basidiomycete fungi. The unique substrate specificity of ScCdc14 was invariant in homologs from eight diverse species of dikarya, suggesting it is conserved across the lineage. A synthetic substrate mimetic inhibited diverse fungal Cdc14 homologs with similar low µMK i values, but had little effect on related phosphatases. Our results justify future exploration of Cdc14 as a broad spectrum antifungal target for plant protection. -
Abstract Protein‐ and peptide‐based therapeutics with high tolerance and specificity along with low off‐target effects and genetic risks have attracted tremendous attention over the last three decades. Herein, a new type of noncationic lipidoid nanoparticle (LNP) is reported for His‐tagged protein delivery. Active lipidoids are synthesized by conjugating a nitrilotriacetic acid group with hydrophobic tails (EC16, O16B, and O17O) and nanoparticles are formulated in the presence of nickel ions and helper lipids (cholesterol, 1,2‐dioleoyl‐
sn ‐glycero‐3‐phosphoethanolamine, and 1,2‐distearoyl‐sn ‐glycero‐3‐phosphoethanolamine‐N ‐[methoxy(polyethylene glycol)‐2000]). It is demonstrated that the newly developed LNPs are capable of delivering various His‐tagged proteins including green fluorescent protein (GFP), (−30)GFP‐Cre recombinase, and CRISPR/Cas9 ribonucleoprotein into mammalian cells. -
null (Ed.)Abstract Mammalian cells process information through coordinated spatiotemporal regulation of proteins. Engineering cellular networks thus relies on efficient tools for regulating protein levels in specific subcellular compartments. To address the need to manipulate the extent and dynamics of protein localization, we developed a platform technology for the target-specific control of protein destination. This platform is based on bifunctional molecules comprising a target-specific nanobody and universal sequences determining target subcellular localization or degradation rate. We demonstrate that nanobody-mediated localization depends on the expression level of the target and the nanobody, and the extent of target subcellular localization can be regulated by combining multiple target-specific nanobodies with distinct localization or degradation sequences. We also show that this platform for nanobody-mediated target localization and degradation can be regulated transcriptionally and integrated within orthogonal genetic circuits to achieve the desired temporal control over spatial regulation of target proteins. The platform reported in this study provides an innovative tool to control protein subcellular localization, which will be useful to investigate protein function and regulate large synthetic gene circuits.more » « less
-
Abstract Numerous methods have been developed in model systems to deplete or inactivate proteins to elucidate their functional roles. In
Caenorhabditis elegans , a common method for protein depletion is RNA interference (RNAi), in which mRNA is targeted for degradation.C. elegans is also a powerful genetic organism, amenable to large‐scale genetic screens and CRISPR‐mediated genome editing. However, these approaches largely lead to constitutive inhibition, which can make it difficult to study proteins essential for development or to dissect dynamic cellular processes. Thus, there have been recent efforts to develop methods to rapidly inactivate or deplete proteins to overcome these barriers. One such method that is proving to be exceptionally powerful is auxin‐inducible degradation. In order to apply this approach inC. elegans , a 44–amino acid degron tag is added to the protein of interest, and theArabidopsis ubiquitin ligase TIR1 is expressed in target tissues. When the plant hormone auxin is added, it mediates an interaction between TIR1 and the degron‐tagged protein of interest, which triggers ubiquitination of the protein and its rapid degradation via the proteasome. Here, we have outlined multiple methods for inducing auxin‐mediated depletion of target proteins inC. elegans , highlighting the versatility and power of this method. © 2021 Wiley Periodicals LLC.This article was corrected on 19 July 2022. See the end of the full text for details.
Basic Protocol 1 : Long‐term auxin‐mediated depletion on platesSupport Protocol : Preparation of NGM and NGM‐auxin platesBasic Protocol 2 : Rapid auxin‐mediated depletion via soakingBasic Protocol 3 : Acute auxin‐mediated depletion in isolated embryosBasic Protocol 4 : Assessing auxin‐mediated depletion -
Abstract Ubiquitin mediated signaling contributes critically to host cell defenses during pathogen infection. Many pathogens manipulate the ubiquitin system to evade these defenses. Here we characterize a likely effector protein bearing a deubiquitylase (DUB) domain from the obligate intracellular bacterium
Orientia tsutsugamushi , the causative agent of scrub typhus. The Ulp1-like DUB prefers ubiquitin substrates over ubiquitin-like proteins and efficiently cleaves polyubiquitin chains of three or more ubiquitins. The co-crystal structure of the DUB (OtDUB) domain with ubiquitin revealed three bound ubiquitins: one engages the S1 site, the second binds an S2 site contributing to chain specificity and the third binds a unique ubiquitin-binding domain (UBD). The UBD modulates OtDUB activity, undergoes a pronounced structural transition upon binding ubiquitin, and binds monoubiquitin with an unprecedented ~5 nM dissociation constant. The characterization and high-resolution structure determination of this enzyme should aid in its development as a drug target to counterOrientia infections.