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Genetics Oct 2023The sleep state is widely observed in animals. The molecular mechanisms underlying sleep regulation, however, remain largely unclear. In the nematode Caenorhabditis...
The sleep state is widely observed in animals. The molecular mechanisms underlying sleep regulation, however, remain largely unclear. In the nematode Caenorhabditis elegans, developmentally timed sleep (DTS) and stress-induced sleep (SIS) are 2 types of quiescent behaviors that fulfill the definition of sleep and share conserved sleep-regulating molecules with mammals. To identify novel sleep-regulating molecules, we conducted an unbiased forward genetic screen based on DTS phenotypes. We isolated 2 mutants, rem8 and rem10, that exhibited significantly disrupted DTS and SIS. The causal gene of the abnormal sleep phenotypes in both mutants was mapped to dgk-1, which encodes diacylglycerol kinase. Perhaps due to the diminished SIS, dgk-1 mutant worms exhibited decreased survival following exposure to a noxious stimulus. Pan-neuronal and/or cholinergic expression of dgk-1 partly rescued the dgk-1 mutant defects in DTS, SIS, and post-stress survival. Moreover, we revealed that pkc-1/nPKC participates in sleep regulation and counteracts the effect of dgk-1; the reduced DTS, SIS, and post-stress survival rate were partly suppressed in the pkc-1; dgk-1 double mutant compared with the dgk-1 single mutant. Excessive sleep observed in the pkc-1 mutant was also suppressed in the pkc-1; dgk-1 double mutant, implying that dgk-1 has a complicated mode of action. Our findings indicate that neuronal DGK-1 is essential for normal sleep and that the counterbalance between DGK-1 and PKC-1 is crucial for regulating sleep and mitigating post-stress damage.
Topics: Animals; Diacylglycerol Kinase; Caenorhabditis elegans; Neurons; Phosphorylation; Sleep; Mammals
PubMed: 37682636
DOI: 10.1093/genetics/iyad140 -
The Journal of Biological Chemistry Sep 2023Mutations in the DNA helicase RECQL4 lead to Rothmund-Thomson syndrome (RTS), a disorder characterized by mitochondrial dysfunctions, premature aging, and genomic...
Mutations in the DNA helicase RECQL4 lead to Rothmund-Thomson syndrome (RTS), a disorder characterized by mitochondrial dysfunctions, premature aging, and genomic instability. However, the mechanisms by which these mutations lead to pathology are unclear. Here we report that RECQL4 is ubiquitylated by a mitochondrial E3 ligase, MITOL, at two lysine residues (K1101, K1154) via K6 linkage. This ubiquitylation hampers the interaction of RECQL4 with mitochondrial importer Tom20, thereby restricting its own entry into mitochondria. We show the RECQL4 2K mutant (where both K1101 and K1154 are mutated) has increased entry into mitochondria and demonstrates enhanced mitochondrial DNA (mtDNA) replication. We observed that the three tested RTS patient mutants were unable to enter the mitochondria and showed decreased mtDNA replication. Furthermore, we found that RECQL4 in RTS patient mutants are hyperubiquitylated by MITOL and form insoluble aggregate-like structures on the outer mitochondrial surface. However, depletion of MITOL allows RECQL4 expressed in these RTS mutants to enter mitochondria and rescue mtDNA replication. Finally, we show increased accumulation of hyperubiquitylated RECQL4 outside the mitochondria leads to the cells being potentiated to increased mitophagy. Hence, we conclude regulating the turnover of RECQL4 by MITOL may have a therapeutic effect in patients with RTS.
Topics: Humans; DNA, Mitochondrial; Mitochondria; Mitophagy; Mutation; RecQ Helicases; Rothmund-Thomson Syndrome; Ubiquitin-Protein Ligases; Ubiquitination; DNA Replication
PubMed: 37495109
DOI: 10.1016/j.jbc.2023.105087 -
Plant Direct Oct 2023The functions of closely related Myb-like repressor and Myb-like activator proteins within the plant circadian oscillator have been well-studied as separate groups, but...
The functions of closely related Myb-like repressor and Myb-like activator proteins within the plant circadian oscillator have been well-studied as separate groups, but the genetic interactions between them are less clear. We hypothesized that these repressors and activators would interact additively to regulate both circadian and growth phenotypes. We used CRISPR-Cas9 to generate new mutant alleles and performed physiological and molecular characterization of plant mutants for five of these core Myb-like clock factors compared with a repressor mutant and an activator mutant. We first examined circadian clock function in plants likely null for both the repressor proteins, () and (), and the activator proteins, (), (), and (). The triple mutant has a long period and flowers late, while quintuple mutants, similarly to mutants, have poor circadian rhythms and flower early. This suggests that and are epistatic to , , and for circadian clock and flowering time function. We next examined hypocotyl elongation and rosette leaf size in these mutants. The mutants have growth phenotypes intermediate between and mutants, suggesting that , , , , and interact additively to regulate growth. Together, our data suggest that these five Myb-like factors interact differently in regulation of the circadian clock versus growth. More generally, the near-norm al seedling phenotypes observed in the largely arrhythmic quintuple mutant demonstrate that circadian-regulated output processes, like control of hypocotyl elongation, do not always depend upon rhythmic oscillator function.
PubMed: 37811362
DOI: 10.1002/pld3.533 -
FEBS Open Bio Aug 2023Wolfram syndrome is a monogenic disease mainly caused by mutations in the WFS1 gene. Mutations in the WFS1 gene give rise to diabetes. Here, we characterized mutant WFS1...
Wolfram syndrome is a monogenic disease mainly caused by mutations in the WFS1 gene. Mutations in the WFS1 gene give rise to diabetes. Here, we characterized mutant WFS1 proteins by studying the stability of full-length wild-type (WT) WFS1, a missense mutant P724L, and two C-terminally truncated mutants, W837X and Y652X. We compared their stability by overexpressing them in MIN6 and HEK293T cells. The C-terminally truncated mutants W837X and Y652X are degraded more rapidly than the missense P724L mutant or wild-type WFS1 in MIN6 cells. In contrast, Y652X is more stable than WT or other mutant WFS1 proteins in HEK293T. In conclusion, we found that C-terminally truncated WFS1 mutants are selectively degraded in a cell type-specific manner.
Topics: Humans; HEK293 Cells; Insulin-Secreting Cells; Mutation; Proteasome Endopeptidase Complex; Wolfram Syndrome
PubMed: 37440664
DOI: 10.1002/2211-5463.13674 -
Archives of Biochemistry and Biophysics Aug 2023The inosine triphosphate pyrophosphatase (ITPA) enzyme plays a critical cellular role by removing noncanonical nucleoside triphosphates from nucleotide pools. One of the...
The inosine triphosphate pyrophosphatase (ITPA) enzyme plays a critical cellular role by removing noncanonical nucleoside triphosphates from nucleotide pools. One of the first pathological ITPA mutants identified is R178C (rs746930990), which causes a fatal infantile encephalopathy, termed developmental and epileptic encephalopathy 35 (DEE 35). The accumulation of noncanonical nucleotides such as inosine triphosphate (ITP), is suspected to affect RNA and/or interfere with normal nucleotide function, leading to development of DEE 35. Molecular dynamics simulations have shown that the very rare R178C mutation does not significantly perturb the overall structure of the protein, but results in a high level of structural flexibility and disrupts active-site hydrogen bond networks, while preliminary biochemical data indicate that ITP hydrolyzing activity is significantly reduced for the R178C mutant. Here we report Michaelis-Menten enzyme kinetics data for the R178C ITPA mutant and three other position 178 ITPA mutants. These data confirm that position 178 is essential for ITPA activity and even conservative mutation at this site (R178K) results in significantly reduced enzyme activity. Our data support that disruption of the active-site hydrogen bond network is a major cause of diminished ITP hydrolyzing activity for the R178C mutation. These results suggest an avenue for developing therapies to address DEE 35.
Topics: Inosine; Pyrophosphatases; Inosine Triphosphate; Arginine; Nucleotides
PubMed: 37506994
DOI: 10.1016/j.abb.2023.109700 -
Scientific Reports Oct 2023Recently, the specific association between Sinonasal inverted papilloma (SIP) and EGFR exon 20 mutations has been reported. To investigate the link between specific EGFR...
Recently, the specific association between Sinonasal inverted papilloma (SIP) and EGFR exon 20 mutations has been reported. To investigate the link between specific EGFR mutations and SIP development, we established organotypic raft culture system using nasal polyp-derived immortalized NP2 (iNP2) cells expressing EGFR exon 20 mutants or an exon 19 mutant, and SIP-derived iIP4 cells harboring P772_H773insPYNP mutation. In the raft culture, iIP4 cells showed the inverted growth pattern characteristic to SIP. Interestingly, iNP2 cells expressing EGFR exon 20 duplication mutants, S768_D770dup and N771_H773dup, but not of EGFR exon 19 mutant, E746_A750del, showed the inverted growth pattern. Enhanced activation of the PI3K/AKT signaling pathway was observed in iNP2_S768_D770dup and iIP4 cells, while increased MAPK signaling was found in iNP2_N771_H773dup. Increased cell migration and invasion were found in all cells carrying EGFR mutations when compared to iNP2 cells, and this effect was inhibited by either PI3K or MEK inhibitor. Notably, iNP2 cells expressing the N771_H773dup mutant showed the highest migration and invasion abilities. These results suggest that specific mutations in EGFR exon 20 play a crucial role in SIP development, partially though hyper-activation of the PI3K/AKT and MAPK signaling pathways. This study presents the first in vitro model for SIP development, which could facilitate further investigations into SIP pathogenesis and preclinical studies for new therapeutic agents.
Topics: Humans; Papilloma, Inverted; ErbB Receptors; Proto-Oncogene Proteins c-akt; Phosphatidylinositol 3-Kinases; Signal Transduction; Head and Neck Neoplasms; Mutation
PubMed: 37891239
DOI: 10.1038/s41598-023-45585-3 -
Applied Microbiology and Biotechnology Nov 2023The ClosTron mutagenesis system has enabled researchers to efficiently edit the clostridial genome. Since site-specific insertion of the mobile ClosTron insert may cause...
The ClosTron mutagenesis system has enabled researchers to efficiently edit the clostridial genome. Since site-specific insertion of the mobile ClosTron insert may cause errors, validation is key. In this paper we describe the use of digital PCR (dPCR) as an alternative tool in selecting clostridial mutant strains. Clostridium perfringens chitinase mutant strains were constructed in which the mobile ClosTron intron was inserted into one of the chitinase genes. On-target insertion of the mobile intron was validated through conventional PCR. In order to confirm the absence of off-target insertions, dPCR was used to determine the amount of the ClosTron intron as well as the amount of a reference gene, located in close proximity to the interrupted gene. Subsequently, mutant strains containing an equivalent amount of both genes were selected as these do not contain additional off-target mobile ClosTron inserts. The outcome of this selection procedure was confirmed through a validated PCR-based approach. In addition to its application in mutant selection, dPCR can be used in other aspects of clostridial research, such as the distinction and easy quantification of different types of strains (wildtype vs. mutant) in complex matrices, such as faecal samples, a process in which other techniques are hampered by bacterial overgrowth (plating) or inhibition by matrix contaminants (qPCR). This research demonstrates that dPCR is indeed a high-throughput method in the selection of clostridial insertion mutants as well as a robust and accurate tool in distinguishing between wildtype and mutant C. perfringens strains, even in a complex matrix such as faeces. KEY POINTS: • Digital PCR as an alternative in ClosTron mutant selection • Digital PCR is an accurate tool in bacterial quantification in a complex matrix • Digital PCR is an alternative tool with great potential to microbiological research.
PubMed: 37704769
DOI: 10.1007/s00253-023-12779-8 -
Fish & Shellfish Immunology Mar 2024Aeromonas hydrophila (A. hydrophila) is a typical zoonotic pathogenic bacterium that infects humans, animals, and fish. It has been reported that the Fur, a Fe...
Aeromonas hydrophila (A. hydrophila) is a typical zoonotic pathogenic bacterium that infects humans, animals, and fish. It has been reported that the Fur, a Fe regulatory protein, and the Crp, a cAMP receptor protein, play important roles in bacterial virulence in many bacteria, but no research has been investigated on A. hydrophila. In this study, the Δfur and Δcrp mutant strains were constructed by the suicide plasmid method. These two mutant strains exhibited a slightly diminished bacterial growth and also were observed some alterations in the number of outer membrane proteins, and the disappearance of hemolysis in the Δcrp strain. Animal experiments of crucian carp showed that the Δfur and Δcrp mutant strains significantly decreased virulence compared to the wild-type strain, and both mutant strains were able to induce good immune responses by two kinds of administration routes of intraperitoneal immunization (i.p) and immersion immunization, and the protection rates through intraperitoneal injection of Δfur and Δcrp to crucian carp were as high as 83.3 % and 73.3 %, respectively, and immersion immunization route of Δfur and Δcrp to crucian carp provided protection as high as 40 % and 20 %, respectively. These two mutant strains showed abilities to induce changes in enzymatic activities of the non-specific enzymes SOD, LZM, AKP, and ACP in crucian carp. Together, these results indicated the Δfur and Δcrp mutants were safe and effective candidate vaccine strains, showing good protection against the wild-type A. hydrophila challenge.
Topics: Humans; Animals; Gram-Negative Bacterial Infections; Vaccines, Attenuated; Aeromonas hydrophila; Fish Diseases; Carps
PubMed: 38244821
DOI: 10.1016/j.fsi.2024.109380 -
G3 (Bethesda, Md.) Dec 2023Rapid and low-cost sequencing, as well as computer analysis, have facilitated the diagnosis of many genetic diseases, resulting in a substantial rise in the number of...
Rapid and low-cost sequencing, as well as computer analysis, have facilitated the diagnosis of many genetic diseases, resulting in a substantial rise in the number of disease-associated genes. However, genetic diagnosis of many disorders remains problematic due to the lack of interpretation for many genetic variants, especially missenses, the infeasibility of high-throughput experiments on mammals, and the shortcomings of computational prediction technologies. Additionally, the available mutant databases are not well-utilized. Toward this end, we used Caenorhabditis elegans mutant resources to delineate the functions of eight missense variants (V444I, V517D, E610K, L732F, E817K, H873P, R1105K, and G1205E) and two stop codons (W937stop and Q1434stop), including several matching variants (MatchVar) with human in ciliopathy associated IFT-140 (also called CHE-11)//IFT140 (intraflagellar transport protein 140). Moreover, MatchVars carrying C. elegans mutants, including IFT-140(G680S) and IFT-140(P702A) for the human (G704S) (dbSNP: rs150745099) and P726A (dbSNP: rs1057518064 and a conflicting variation) were created using CRISPR/Cas9. IFT140 is a key component of IFT complex A (IFT-A), which is involved in the retrograde transport of IFT along cilia and the entrance of G protein-coupled receptors into cilia. Functional analysis of all 10 variants revealed that P702A and W937stop, but not others phenocopied the ciliary phenotypes (short cilia, IFT accumulations, mislocalization of membrane proteins, and cilia entry of nonciliary proteins) of the IFT-140 null mutant, indicating that both P702A and W937stop are phenotypic in C. elegans. Our functional data offered experimental support for interpreting human variants, by using ready-to-use mutants carrying MatchVars and generating MatchVars with CRISPR/Cas9.
Topics: Animals; Humans; Caenorhabditis elegans; Flagella; Cilia; Biological Transport; Caenorhabditis elegans Proteins; Mammals
PubMed: 37933433
DOI: 10.1093/g3journal/jkad227 -
Cold Spring Harbor Protocols Nov 2023The starting point in a mutational analysis of gene function is obtaining or producing a mutant. Here different methods of obtaining mouse mutants are discussed,...
The starting point in a mutational analysis of gene function is obtaining or producing a mutant. Here different methods of obtaining mouse mutants are discussed, including screening for spontaneous mutants, screening for mutants following chemical or X-ray mutagenesis, and producing mutations through targeted manipulation of the genome. Manipulation of the genome can be random, as in different types of insertional mutagenesis. Alternatively, targeted manipulation such as gene targeting using homologous recombination in embryonic stem (ES) cells or gene editing by CRISPR-Cas can be used to produce custom mutations in a specific gene. The basic methods are outlined, and the advantages and disadvantages of homologous recombination and CRISPR-Cas gene editing are discussed. Resources for obtaining mutations that already exist are provided. If, for your planned study, no suitable mutations are available, there is advice about what you should know about your gene of interest before embarking on a gene targeting experiment.
PubMed: 37932077
DOI: 10.1101/pdb.over107956