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BMC Ecology and Evolution Oct 2023Cyclic di-guanylate (c-di-GMP), synthesized by diguanylate cyclase, is a major second messenger in prokaryotes, where it triggers biofilm formation. The dictyostelid...
BACKGROUND
Cyclic di-guanylate (c-di-GMP), synthesized by diguanylate cyclase, is a major second messenger in prokaryotes, where it triggers biofilm formation. The dictyostelid social amoebas acquired diguanylate cyclase (dgcA) by horizontal gene transfer. Dictyostelium discoideum (Ddis) in taxon group 4 uses c-di-GMP as a secreted signal to induce differentiation of stalk cells, the ancestral somatic cell type that supports the propagating spores. We here investigated how this role for c-di-GMP evolved in Dictyostelia by exploring dgcA function in the group 2 species Polysphondylium pallidum (Ppal) and in Polysphondylium violaceum (Pvio), which resides in a small sister clade to group 4.
RESULTS
Similar to Ddis, dgcA is upregulated after aggregation in Ppal and Pvio and predominantly expressed in the anterior region and stalks of emerging fruiting bodies. DgcA null mutants in Ppal and Pvio made fruiting bodies with very long and thin stalks and only few spores and showed delayed aggregation and larger aggregates, respectively. Ddis dgcA- cells cannot form stalks at all, but showed no aggregation defects. The long, thin stalks of Ppal and Pvio dgcA- mutants were also observed in acaA- mutants in these species. AcaA encodes adenylate cyclase A, which mediates the effects of c-di-GMP on stalk induction in Ddis. Other factors that promote stalk formation in Ddis are DIF-1, produced by the polyketide synthase StlB, low ammonia, facilitated by the ammonia transporter AmtC, and high oxygen, detected by the oxygen sensor PhyA (prolyl 4-hydroxylase). We deleted the single stlB, amtC and phyA genes in Pvio wild-type and dgcA- cells. Neither of these interventions affected stalk formation in Pvio wild-type and not or very mildly exacerbated the long thin stalk phenotype of Pvio dgcA- cells.
CONCLUSIONS
The study reveals a novel role for c-di-GMP in aggregation, while the reduced spore number in Pvio and Ppal dgcA- is likely an indirect effect, due to depletion of the cell pool by the extended stalk formation. The results indicate that in addition to c-di-GMP, Dictyostelia ancestrally used an as yet unknown factor for induction of stalk formation. The activation of AcaA by c-di-GMP is likely conserved throughout Dictyostelia.
Topics: Dictyostelium; Ammonia; Phosphorus-Oxygen Lyases; Dictyosteliida; Oxygen
PubMed: 37803310
DOI: 10.1186/s12862-023-02169-z -
Current Biology : CB Aug 2023Macropinocytosis is a conserved endocytic process by which cells engulf droplets of medium into micron-sized vesicles. We use light-sheet microscopy to define an...
Macropinocytosis is a conserved endocytic process by which cells engulf droplets of medium into micron-sized vesicles. We use light-sheet microscopy to define an underlying set of principles by which macropinocytic cups are shaped and closed in Dictyostelium amoebae. Cups form around domains of PIP3 stretching almost to their lip and are supported by a specialized F-actin scaffold from lip to base. They are shaped by a ring of actin polymerization created by recruiting Scar/WAVE and Arp2/3 around PIP3 domains, but how cups evolve over time to close and form a vesicle is unknown. Custom 3D analysis shows that PIP3 domains expand from small origins, capturing new membrane into the cup, and crucially, that cups close when domain expansion stalls. We show that cups can close in two ways: either at the lip, by inwardly directed actin polymerization, or the base, by stretching and delamination of the membrane. This provides the basis for a conceptual mechanism whereby closure is brought about by a combination of stalled cup expansion, continued actin polymerization at the lip, and membrane tension. We test this through the use of a biophysical model, which can recapitulate both forms of cup closure and explain how 3D cup structures evolve over time to mediate engulfment.
Topics: Actins; Dictyostelium; Cell Membrane Structures; Actin Cytoskeleton; Endocytosis
PubMed: 37379843
DOI: 10.1016/j.cub.2023.06.017 -
International Journal of Molecular... Sep 2023is the primary causative agent of Legionnaires' disease. The mutant-type strain interrupted in the ORF7 gene region responsible for the lipopolysaccharide biosynthesis...
is the primary causative agent of Legionnaires' disease. The mutant-type strain interrupted in the ORF7 gene region responsible for the lipopolysaccharide biosynthesis of the strain Heysham-1, lacking the -acetyl groups attached to the rhamnose of the core part, showed a higher surface polarity compared with the wild-type strain. The measurement of excitation energy transfer between fluorophores located on the surface of bacteria and eukaryotic cells showed that, at an early stage of interaction with host cells, the mutant exhibited weaker interactions with cells and THP-1-derived macrophages. The mutant displayed reduced adherence to macrophages but enhanced adherence to , suggesting that the -acetyl group of the LPS core region plays a crucial role in facilitating interaction with macrophages. The lack of core rhamnose -acetyl groups made it easier for the bacteria to multiply in amoebae and macrophages. The mutant induced TNF-α production more strongly compared with the wild-type strain. The mutant synthesized twice as many ceramides Cer(t34:0) and Cer(t38:0) than the wild-type strain. The study showed that the internal sugars of the LPS core region of sg 1 can interact with eukaryotic cell surface receptors and mediate in contacting and attaching bacteria to host cells as well as modulating the immune response to infection.
Topics: Humans; Legionella pneumophila; Lipopolysaccharides; Rhamnose; Serogroup; Bacterial Proteins; Legionnaires' Disease
PubMed: 37834049
DOI: 10.3390/ijms241914602 -
RNA (New York, N.Y.) Nov 2023The mammalian mRNA 5' cap structures play important roles in cellular processes such as nuclear export, efficient translation, and evading cellular innate immune...
The mammalian mRNA 5' cap structures play important roles in cellular processes such as nuclear export, efficient translation, and evading cellular innate immune surveillance and regulating 5'-mediated mRNA turnover. Hence, installation of the proper 5' cap is crucial in therapeutic applications of synthetic mRNA. The core 5' cap structure, Cap-0, is generated by three sequential enzymatic activities: RNA 5' triphosphatase, RNA guanylyltransferase, and cap N7-guanine methyltransferase. Vaccinia virus RNA capping enzyme (VCE) is a heterodimeric enzyme that has been widely used in synthetic mRNA research and manufacturing. The large subunit of VCE D1R exhibits a modular structure where each of the three structural domains possesses one of the three enzyme activities, whereas the small subunit D12L is required to activate the N7-guanine methyltransferase activity. Here, we report the characterization of a single-subunit RNA capping enzyme from an amoeba giant virus. Faustovirus RNA capping enzyme (FCE) exhibits a modular array of catalytic domains in common with VCE and is highly efficient in generating the Cap-0 structure without an activation subunit. Phylogenetic analysis suggests that FCE and VCE are descended from a common ancestral capping enzyme. We found that compared to VCE, FCE exhibits higher specific activity, higher activity toward RNA containing secondary structures and a free 5' end, and a broader temperature range, properties favorable for synthetic mRNA manufacturing workflows.
Topics: Animals; Phylogeny; RNA, Messenger; Nucleotidyltransferases; RNA; Methyltransferases; Guanine; RNA Caps; Mammals
PubMed: 37625853
DOI: 10.1261/rna.079738.123 -
Biomolecules Aug 2023The proteasome, a complex multi-catalytic protease machinery, orchestrates the protein degradation essential for maintaining cellular homeostasis, and its dysregulation... (Review)
Review
The proteasome, a complex multi-catalytic protease machinery, orchestrates the protein degradation essential for maintaining cellular homeostasis, and its dysregulation also underlies many different types of diseases. Its function is regulated by many different mechanisms that encompass various factors such as proteasome activators (PAs), adaptor proteins, and post-translational modifications. This review highlights the unique characteristics of proteasomal regulation through the lens of a distinct family of regulators, the 11S, REGs, or PA26/PA28. This ATP-independent family, spanning from amoebas to mammals, exhibits a common architectural structure; yet, their cellular biology and criteria for protein degradation remain mostly elusive. We delve into their evolution and cellular biology, and contrast their structure and function comprehensively, emphasizing the unanswered questions regarding their regulatory mechanisms and broader roles in proteostasis. A deeper understanding of these processes will illuminate the roles of this regulatory family in biology and disease, thus contributing to the advancement of therapeutic strategies.
Topics: Animals; Proteasome Endopeptidase Complex; Allosteric Regulation; Cytoplasm; Mammals; Proteolysis
PubMed: 37759726
DOI: 10.3390/biom13091326 -
Journal of Chemical Theory and... Nov 2023Understanding cooperativity and frustration is crucial for studying biological processes such as molecular recognition and protein aggregation. Force fields have been...
Understanding cooperativity and frustration is crucial for studying biological processes such as molecular recognition and protein aggregation. Force fields have been extensively utilized to explore cooperativity in the formation of protein secondary structures and self-assembled systems. Multiple studies have demonstrated that polarizable force fields provide more accurate descriptions of this phenomenon compared to fixed-charge pairwise nonpolarizable force fields, thanks to the incorporation of polarization effects. In this study, we assess the performance of the AMOEBA polarizable force field and the AMBER and OPLS nonpolarizable pairwise force fields in capturing positive and negative cooperativity recently explored in neutral and charged molecular clusters using density functional theory. Our findings show that polarizable and nonpolarizable force fields qualitatively reproduce the relative cooperativity observed in electron structure calculations. However, AMBER and OPLS fail to describe absolute cooperativity. In contrast, AMOEBA accounts for the absolute cooperativity by considering interactions beyond pairwise interactions. According to the energy decomposition analysis, it is observed that the electrostatic interactions calculated with the AMBER and OPLS force fields seem to play an important and counterintuitive role in reproducing the adiabatic interaction energies calculated with density functional theory. However, it is important to note that these force fields, due to their nature, do not explicitly incorporate many-body effects, which limits their ability to accurately describe cooperativity. On the other hand, frustration in polarizable and nonpolarizable force fields is caused by changes in bond stretching and angle bending terms of the building blocks when they are forming a complex.
PubMed: 37888874
DOI: 10.1021/acs.jctc.3c00762 -
Journal of Biomolecular Structure &... Aug 2023Cholesterol is one of the essential component of lipid in membrane. We present a polarizable atomic multipole force field (FF) for the molecular dynamic simulation of...
Cholesterol is one of the essential component of lipid in membrane. We present a polarizable atomic multipole force field (FF) for the molecular dynamic simulation of cholesterol. The FF building process follows the computational framework as the atomic multipole optimized energetics for biomolecular applications (AMOEBA) model. In this framework, the electronics parameters, including atomic monopole moments, dipole moments, and quadrupole moments calculated from ab initio calculations in the gas phase, are applied to represent the charge distribution. Furthermore, the many-body polarization is modeled by following the same pattern of distributed atomic polarizabilities. Then, the bilayers composed of two typical phospholipid molecules, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), in a range of different cholesterol concentrations are built and implemented by molecular dynamics (MD) simulations based on the proposed polarizable FF. The simulation results are statistically analyzed to validate the feasibility of the proposed FF. The structural properties of the bilayers are calculated to compare with the related experimental values. The MD values show the same trend of experimental values changes.Communicated by Ramaswamy H. Sarma.
PubMed: 37565356
DOI: 10.1080/07391102.2023.2245045 -
Molecular and Biochemical Parasitology Dec 2023Acanthamoeba are known to cause a vision threatening eye infection typically due to contact lens wear, and an infection of the central nervous system. The ability of...
Acanthamoeba are known to cause a vision threatening eye infection typically due to contact lens wear, and an infection of the central nervous system. The ability of these amoebae to switch phenotypes, from an active trophozoite to a resistant cyst form is not well understood; the cyst stage is often resistant to chemotherapy, which is of concern given the rise of contact lens use and the ineffective disinfectants available, versus the cyst stage. Herein, for the first time, a range of raloxifene sulfonate/sulfamate derivatives which target nucleotide pyrophosphatase/phosphodiesterase enzymes, were assessed using amoebicidal and excystation tests versus the trophozoite and cyst stage of Acanthamoeba. Moreover, the potential for cytopathogenicity inhibition in amoebae was assessed. Each of the derivatives showed considerable anti-amoebic activity as well as the ability to suppress phenotypic switching (except for compound 1a). Selected raloxifene derivatives reduced Acanthamoeba-mediated host cell damage using lactate dehydrogenase assay. These findings suggest that pyrophosphatase/phosphodiesterase enzymes may be valuable targets against Acanthamoeba infections.
Topics: Animals; Acanthamoeba castellanii; Raloxifene Hydrochloride; Sulfonic Acids; Trophozoites; Alkanesulfonates; Phosphoric Diester Hydrolases
PubMed: 37562558
DOI: 10.1016/j.molbiopara.2023.111582 -
Ecological Applications : a Publication... Sep 2023In fire-prone ecosystems, knowledge of vegetation-fire-climate relationships and the history of fire suppression and Indigenous cultural burning can inform discussions...
In fire-prone ecosystems, knowledge of vegetation-fire-climate relationships and the history of fire suppression and Indigenous cultural burning can inform discussions of how to use fire as a management tool, particularly as climate continues to change rapidly. On Wiisaakodewan-minis/Stockton Island in the Apostle Islands National Lakeshore of Wisconsin, USA, structural changes in a pine-dominated natural area containing a globally rare barrens community occurred after the cessation of cultural burning by the Indigenous Ojibwe people and the imposition of fire-suppression policies, leading to questions about the historical role of fire in this culturally and ecologically important area. To help understand better the ecological context needed to steward these pine forest and barrens communities, we developed palaeoecological records of vegetation, fire, and hydrological change using pollen, charcoal, and testate amoebae preserved in peat and sediment cores collected from bog and lagoon sediments within the pine-dominated landscape. Results indicated that fire has been an integral part of Stockton Island ecology for at least 6000 years. Logging in the early 1900s led to persistent changes in island vegetation, and post-logging fires of the 1920s and 1930s were anomalous in the context of the past millennium, likely reflecting more severe and/or extensive burning than in the past. Before that, the composition and structure of pine forest and barrens had changed little, perhaps due to regular low-severity surface fires, which may have occurred with a frequency consistent with Indigenous oral histories (~4-8 years). Higher severity fire episodes, indicated by large charcoal peaks above background levels in the records, occurred predominantly during droughts, suggesting that more frequent or more intense droughts in the future may increase fire frequency and severity. The persistence of pine forest and barrens vegetation through past periods of climatic change indicates considerable ecological resistance and resilience. Future persistence in the face of climate changes outside this historical range of variability may depend in part on returning fire to these systems.
Topics: Humans; Ecosystem; Charcoal; Forests; Fires; Wisconsin; Pinus; Trees
PubMed: 37334723
DOI: 10.1002/eap.2901 -
Biological Reviews of the Cambridge... Dec 2023Multicellularity evolved multiple times in the history of life, with most reviewers agreeing that it appeared at least 20 times in eukaryotes. However, a specific list... (Review)
Review
Multicellularity evolved multiple times in the history of life, with most reviewers agreeing that it appeared at least 20 times in eukaryotes. However, a specific list of multicellular eukaryotes with clear criteria for inclusion has not yet been published. Herein, an updated critical review of eukaryotic multicellularity is presented, based on current understanding of eukaryotic phylogeny and new discoveries in microbiology, phycology and mycology. As a result, 45 independent multicellular lineages are identified that fall into six distinct types. Functional criteria, as distinct from a purely topological definition of a cell, are introduced to bring uniformity and clarity to the existing definitions of terms such as colony, multicellularity, thallus or plasmodium. The category of clonal multicellularity is expanded to include: (i) septated multinucleated thalli found in Pseudofungi and early-branching Fungi such as Chytridiomycota and Blastocladiomycota; and (ii) multicellular reproductive structures formed by plasmotomy in intracellular parasites such as Phytomyxea. Furthermore, (iii) endogeneous budding, as found in Paramyxida, is described as a form of multicellularity. The best-known case of clonal multicellularity, i.e. (iv) non-separation of cells after cell division, as known from Metazoa and Ochrophyta, is also discussed. The category of aggregative multicellularity is expanded to include not only (v) pseudoplasmodial forms, such a sorocarp-forming Acrasida, but also (vi) meroplasmodial organisms, such as members of Variosea or Filoreta. A common set of topological, geometric, genetic and life-cycle criteria are presented that form a coherent, philosophically sound framework for discussing multicellularity. A possibility of a seventh type of multicellularity is discussed, that of multi-species superorganisms formed by protists with obligatory bacterial symbionts, such as some members of Oxymonada or Parabasalia. Its inclusion is dependent on the philosophical stance taken towards the concepts of individuality and organism in biology. Taxa that merit special attention are identified, such as colonial Centrohelea, and a new speculative form of multicellularity, possibly present in some reticulopodial amoebae, is briefly described. Because of insufficient phylogenetic and morphological data, not all lineages could be unequivocally identified, and the true total number of all multicellular eukaryotic lineages is therefore higher, likely close to a hundred.
Topics: Phylogeny; Eukaryota; Fungi; Biological Evolution
PubMed: 37475165
DOI: 10.1111/brv.13001