Lysine acetylation is a common protein post-translational modification in bacteria and eukaryotes. Unlike phosphorylation, whose functional role in signaling has been established, it is unclear what regulatory mechanism acetylation plays and whether it is conserved across evolution. By performing a proteomic analysis of 48 phylogenetically distant bacteria, we discovered conserved acetylation sites on catalytically essential lysine residues that are invariant throughout evolution. Lysine acetylation removes the residue’s charge and changes the shape of the pocket required for substrate or cofactor binding. Two-thirds of glycolytic and tricarboxylic acid (TCA) cycle enzymes are acetylated at these critical sites. Our data suggest that acetylation may play a direct role in metabolic regulation by switching off enzyme activity. We propose that protein acetylation is an ancient and widespread mechanism of protein activity regulation.
The process by which nonenveloped viruses cross cell membranes during host cell entry remains poorly defined; however, common themes are emerging. Here, we use correlated in vivo and in vitro studies to understand the mechanism of Flock House virus (FHV) entry and membrane penetration. We demonstrate that low endocytic pH is required for FHV infection, that exposure to acidic pH promotes FHV-mediated disruption of model membranes (liposomes), and particles exposed to low pH in vitro exhibit increased hydrophobicity. In addition, FHV particles perturbed by heating displayed a marked increase in liposome disruption, indicating that membrane-active regions of the capsid are exposed or released under these conditions. We also provide evidence that autoproteolytic cleavage, to generate the lipophilic ␥ peptide (4.4 kDa), is required for membrane penetration. Mutant, cleavage-defective particles failed to mediate liposome lysis, regardless of pH or heat treatment, suggesting that these particles are not able to expose or release the requisite membrane-active regions of the capsid, namely, the ␥ peptides. Based on these results, we propose an updated model for FHV entry in which (i) the virus enters the host cell by endocytosis, (ii) low pH within the endocytic pathway triggers the irreversible exposure or release of ␥ peptides from the virus particle, and (iii) the exposed/released ␥ peptides disrupt the endosomal membrane, facilitating translocation of viral RNA into the cytoplasm.Flock House virus (FHV), a nonenveloped, positive-sense RNA virus, has been employed as a model system in several important studies to address a wide range of biological questions (reviewed in reference 55). FHV has been instrumental in understanding virus structure and assembly (17,19,45), RNA replication (2,3,37), and specific packaging of the genome (33,44,53,54). Studies of FHV infection in Drosophila melanogaster flies have provided valuable information about the antiviral innate immune response in invertebrate hosts (29,57). FHV is also used in nanotechnology applications as an epitope-presenting platform to develop novel vaccines and medical therapies (31,48). In this report, we use FHV as a model system to further elucidate the means by which nonenveloped viruses enter host cells and traverse cellular membranes.
Saccharomyces boulardii is a probiotic yeast that has been used for promoting gut health as well as preventing diarrheal diseases. This yeast not only exhibits beneficial phenotypes for gut health but also can stay longer in the gut than Saccharomyces cerevisiae. Therefore, S. boulardii is an attractive host for metabolic engineering to produce biomolecules of interest in the gut. However, the lack of auxotrophic strains with defined genetic backgrounds has hampered the use of this strain for metabolic engineering. Here, we report the development of well-defined auxotrophic mutants (leu2, ura3, his3, and trp1) through clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9-based genome editing. The resulting auxotrophic mutants can be used as a host for introducing various genetic perturbations, such as overexpression or deletion of a target gene, using existing genetic tools for S. cerevisiae. We demonstrated the overexpression of a heterologous gene (lacZ), the correct localization of a target protein (red fluorescent protein) into mitochondria by using a protein localization signal, and the introduction of a heterologous metabolic pathway (xylose-assimilating pathway) in the genome of S. boulardii. We further demonstrated that human lysozyme, which is beneficial for human gut health, could be secreted by S. boulardii. Our results suggest that more sophisticated genetic perturbations to improve S. boulardii can be performed without using a drug resistance marker, which is a prerequisite for in vivo applications using engineered S. boulardii.
Recent studies have established that several nonenveloped viruses utilize virus-encoded lytic peptides for host membrane disruption. We investigated this mechanism with the "gamma" peptide of the insect virus Flock House virus (FHV). We demonstrate that the C terminus of gamma is essential for membrane disruption in vitro and the rescue of immature virus infectivity in vivo, and the amphipathic N terminus of gamma alone is not sufficient. We also show that deletion of the C-terminal domain disrupts icosahedral ordering of the amphipathic helices of gamma in the virus. Our results have broad implications for understanding membrane lysis during nonenveloped virus entry.The presence of membrane lytic peptides in many nonenveloped viruses is well established (3, 16), but how these peptides are deployed from the virus capsid during host cell entry and disrupt membranes remains unclear. These peptides are typically generated by a postassembly proteolytic processing event (1, 11) and are exposed from a previously buried position during conformational alterations in the capsid triggered by host cell conditions (2, 18). Flock House virus (FHV), an insect nodavirus, contains a 4-kDa peptide called "gamma" (␥), which shares many of the characteristics of other nonenveloped virus lytic peptides (3). The FHV capsid is made from 180 copies of a single-coat protein (␣) enclosing a single-stranded bipartite RNA genome (9). Gamma is generated by the autocatalytic cleavage of ␣ during virus maturation (␣ 3  ϩ ␥) (15), remains localized in the capsid interior (9) with occasional externalization or "breathing" (6), and is exposed under low-pH conditions in the endosomes during entry (Odegard et al., submitted for publication). Covalently independent gamma is necessary for virus infection, since maturation-defective FHV (D75N/N363T FHV), which does not undergo the autocleavage of ␣, is not infectious (15,17). The N-terminal ϳ21 residues of gamma (corresponding to residues 364 to 384 of ␣) constitute an amphipathic helix which can disrupt membranes in vitro when synthetically produced (4, 5) and is recognized as the host membrane-interacting region of FHV during entry. The hydrophobic, ϳ23-residue-long C terminus of gamma, especially certain phenylalanine residues (at positions 402, 405, and 407), is responsible for specifically packaging viral RNA into capsids during assembly (14).It was recently demonstrated that a supply of full-length gamma from noninfectious virus-like particles (VLPs) of FHV (13) during entry can restore infectivity to maturation-defective FHV (17), suggesting that gamma can function in trans to mediate access into host cells. This trans-complementation assay (17) was utilized to provide a quantitative readout of the effect of gamma mutations specifically on virus entry and to thus assess the region(s) of gamma required during virus entry. To determine the minimal sequence of gamma required for trans-complementation, FHV VLPs were produced that included the first 384, 390, or 395 amino acids of capsid protein ...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.