Nocturnin in the demosponge Suberites domuncula: a potential circadian clock protein controlling glycogenin synthesis in sponges

2012 ◽  
Vol 448 (2) ◽  
pp. 233-242 ◽  
Author(s):  
Werner E. G. Müller ◽  
Xiaohong Wang ◽  
Vlad A. Grebenjuk ◽  
Michael Korzhev ◽  
Matthias Wiens ◽  
...  
Keyword(s):  
Circadian Clock ◽  
Sequence Similarity ◽  
Three Dimensional ◽  
Metabolic Enzyme ◽  
High Sequence Similarity ◽  
Suberites Domuncula ◽  
Light Supply ◽  
The Stability ◽  
Clock Protein

Sponges are filter feeders that consume a large amount of energy to allow a controlled filtration of water through their aquiferous canal systems. It has been shown that primmorphs, three-dimensional cell aggregates prepared from the demosponge Suberites domuncula and cultured in vitro, change their morphology depending on the light supply. Upon exposure to light, primmorphs show a faster and stronger increase in DNA, protein and glycogen content compared with primmorphs that remain in the dark. The sponge genome contains nocturnin, a light/dark-controlled clock gene, the protein of which shares a high sequence similarity with the related molecule of higher metazoans. The sponge nocturnin protein was found showing a poly(A)-specific 3′-exoribonuclease activity. In addition, the cDNA of the glycogenin gene was identified for subsequent expression studies. Antibodies against nocturnin were raised and used in parallel with the cDNA to determine the regional expression of nocturnin in intact sponge specimens; the highest expression of nocturnin was seen in the epithelial layer around the aquiferous canals. Quantitative PCR analyses revealed that primmorphs after transfer from light to dark show a 10-fold increased expression in the nocturnin gene. In contrast, the expression level of glycogenin decreases in the dark by 3– 4-fold. Exposure of primmorphs to light causes a decrease in nocturnin transcripts and a concurrent increase in glycogenin transcripts. It was concluded that sponges are provided with the molecular circadian clock protein nocturnin that is highly expressed in the dark where it controls the stability of a key metabolic enzyme, glycogenin.

Degradation of the Neurospora circadian clock protein FREQUENCY through the ubiquitin–proteasome pathway

2005 ◽  
Vol 33 (5) ◽  
pp. 953-956 ◽  
Author(s):  
Q. He ◽  
Y. Liu
Keyword(s):  
Circadian Clock ◽  
Major Function ◽  
Ubiquitin Proteasome Pathway ◽  
Mutant Strains ◽  
Ubiquitin Proteasome ◽  
Proteasome Pathway ◽  
The Stability ◽  
Clock Protein

Phosphorylation of the Neurospora circadian clock protein FREQUENCY (FRQ) promotes its degradation through the ubiquitin–proteasome pathway. Ubiquitination of FRQ requires FWD-1 (F-box/WD-40 repeat-containing protein-1), which is the substrate-recruiting subunit of an SCF (SKP/Cullin/F-box)-type ubiquitin ligase. In the fwd-1 mutant strains, FRQ degradation is defective, resulting in the accumulation of hyperphosphorylated FRQ and the loss of the circadian rhythmicities. The CSN (COP9 signalosome) promotes the function of SCF complexes in vivo. But in vitro, deneddylation of cullins by CSN inhibits SCF activity. In Neurospora, the disruption of the csn-2 subunit impairs FRQ degradation and compromises the normal circadian functions. These defects are due to the dramatically reduced levels of FWD-1 in the csn-2 mutant, a result of its rapid degradation. Other components of the SCFFWD−1 complex, SKP-1 and CUL-1 are also unstable in the mutant. These results establish important roles for SCFFWD−1 and CSN in the circadian clock of Neurospora and suggest that they are conserved components of the eukaryotic circadian clocks. In addition, these findings resolve the CSN paradox and suggest that the major function of CSN is to maintain the stability of SCF ubiquitin ligases in vivo.

scholarly journals A Novel Regulatory Role for the Circadian Clock Protein TOC1 via RNA binding

2020 ◽  
Author(s):  
Yi Li ◽  
Yong-Gang Chang ◽  
Dawn H. Nagel ◽  
Tingjian Chen ◽  
Matias L Rugnone ◽  
...  
Keyword(s):  
Circadian Clock ◽  
Rna Binding ◽  
Mutational Analysis ◽  
Sequence Similarity ◽  
Transcriptional Repressor ◽  
Titration Calorimetry ◽  
Mobility Shift ◽  
Rna Motif ◽  
Clock Protein

AbstractThe circadian clock enables plants to predict daily changes of external signals and synchronize them with internal processes, conferring enhanced fitness and growth vigor. The first described Arabidopsis circadian clock protein is TIMING OF CAB EXPRESSION 1 (TOC1), which functions in a transcriptional feedback loop with two myb transcription factors, CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY). Previous studies have shown that TOC1 is a DNA-binding transcriptional repressor of CCA1 and LHY. However, the DNA motifs enriched amongst TOC1 targets share weak sequence similarity and lack consensus, suggesting that TOC1 regulates the expression of its targets through a novel mechanism. Here we show that the TOC1 protein binds directly to RNA via its conserved CCT domain. Using in vitro RNA selection, we identified an RNA motif that is recognized by the TOC1-CCT domain. The TOC1-CCT domain binds to this RNA sequence with nanomolar affinity determined by quantitative electrophoretic mobility shift assays (EMSAs) and isothermal titration calorimetry (ITC). NMR experiments showed that two CCT fragments, CCT533-547 and CCT550-565, use basic residues to bind the RNA motif. Mutational analysis confirmed that lysyl and arginyl residues bind to RNA in a cooperative manner. Furthermore, transiently expressed wildtype and mutant TOC1 in protoplasts demonstrated that RNA binding activity of TOC1 is required for its function as a transcriptional repressor in vivo. Our results reveal a novel regulatory mechanism for TOC1 through RNA binding, suggesting that TOC1 might play key roles as a multi-function protein.

Evolution Analysis of the Circadian Clock Protein KaiB

2013 ◽  
Vol 647 ◽  
pp. 391-395
Author(s):  
Liu Sen ◽  
Song Liu
Keyword(s):  
Circadian Rhythms ◽  
Circadian Clock ◽  
Feedback Loop ◽  
Circadian System ◽  
Circadian Rhythmicity ◽  
Physiological Functions ◽  
Clock System ◽  
Evolution Analysis ◽  
Clock Protein

Regulation of daily physiological functions with approximate a 24-hour periodicity, or circadian rhythms, is a characteristic of eukaryotes. So far, cyanobacteria are only known prokaryotes reported to possess circadian rhythmicity. The circadian system in cyanobacteria comprises both a post-translational oscillator (PTO) and a transcriptional/translational feedback loop (TTFL). The PTO can be reconstituted in vitro with three purified proteins (KaiA, KaiB, and KaiC) with the existence of ATP. Phase of the nanoclockwork has been associated with the phosphorylation states of KaiC, with KaiA promoting the phosphorylation of KaiC, and KaiB de-phosphorylating KaiC. Here we studied the evolution of the KaiB protein. The result will be helpful in understanding the evolution of the circadian clock system.

scholarly journals Circadian Clock Protein PERIOD2 Suppresses the PI3K/Akt Pathway and Promotes Cisplatin Sensitivity in Ovarian Cancer

2020 ◽  
Author(s):  
Zhaoxia Wang ◽  
Fengyan Li ◽  
Meiyan Wei ◽  
Sanyuan Zhang ◽  
Tong Wang
Keyword(s):  
Ovarian Cancer ◽  
Circadian Clock ◽  
Caspase 3 ◽  
Inflammatory Responses ◽  
Akt Pathway ◽  
Tumor Tissues ◽  
Skov3 Cells ◽  
Clock Protein ◽  
E Cadherin

Abstract Background Circadian clock protein PERIOD2 (PER2) acts as a tumor suppressor in cancer; however, little is known about its involvement in chemosensitivity. Methods This study aimed to investigate the role and underlying mechanisms of PER2 in ovarian cancer sensitivity to cisplatin. Overexpression and knockdown of PER2 were performed to explore its role in ovarian cancer cell sensitivity to cisplatin both in vitro and in vivo. The protein levels of PI3K, AKT, caspase 3, E-cadherin, and other drug resistance-related molecules were determined in parental SKOV3 and SKOV3/DDP cells as well as in xenograft tumor tissues. Results Compared with parental cells, SKOV3/DDP cells had dramatically decreased PER2 expression, possibly due to hypermethylation in the PER2 promoter. PER2 overexpression significantly inhibited proliferation while promoting cisplatin-induced apoptosis in SKOV3 and SKOV3/DDP cells. In agreement, PER2-overexpressing SKOV3/DPP cells yielded significantly reduced tumor mass in cisplatin-treated mice compared with control cells. Mechanistically, PER2 overexpression remarkably reduced the protein amounts of PI3K, AKT, and MDR1, while increasing those of caspase 3 and E-cadherin in tumor tissues. Knockdown of PER2 exhibited opposite effects. PER2 overexpression also reduced the serum levels of TNF-α and IL-6 in tumor-bearing mice before the initiation of cisplatin treatment. Conclusion This study suggests that loss of PER2 contributes to cisplatin resistance in SKOV3 cells, possibly by activating the PI3K/AKT pathway and EMT, inhibiting apoptosis, and promoting drug efflux and inflammatory responses. Overexpression of PER2 could reverse these alterations and sensitize both parental SKOV3 and SKOV3/DDP cells to cisplatin.

scholarly journals Insights into Transcriptional Repression of the Homologous Toxin-Antitoxin Cassettes yefM-yoeB and axe-txe

2020 ◽  
Vol 21 (23) ◽  
pp. 9062
Author(s):  
Barbara Kędzierska ◽  
Katarzyna Potrykus ◽  
Agnieszka Szalewska-Pałasz ◽  
Beata Wodzikowska
Keyword(s):  
Transcriptional Repression ◽  
Transcription Initiation ◽  
Sequence Similarity ◽  
In Vitro Transcription ◽  
High Sequence Similarity ◽  
Sequence Composition ◽  
Repressor Complex ◽  
Transcriptional Fusions

Transcriptional repression is a mechanism which enables effective gene expression switch off. The activity of most of type II toxin-antitoxin (TA) cassettes is controlled in this way. These cassettes undergo negative autoregulation by the TA protein complex which binds to the promoter/operator sequence and blocks transcription initiation of the TA operon. Precise and tight control of this process is vital to avoid uncontrolled expression of the toxin component. Here, we employed a series of in vivo and in vitro experiments to establish the molecular basis for previously observed differences in transcriptional activity and repression levels of the pyy and pat promoters which control expression of two homologous TA systems, YefM-YoeB and Axe-Txe, respectively. Transcriptional fusions of promoters with a lux reporter, together with in vitro transcription, EMSA and footprinting assays revealed that: (1) the different sequence composition of the −35 promoter element is responsible for substantial divergence in strengths of the promoters; (2) variations in repression result from the TA repressor complex acting at different steps in the transcription initiation process; (3) transcription from an additional promoter upstream of pat also contributes to the observed inefficient repression of axe-txe module. This study provides evidence that even closely related TA cassettes with high sequence similarity in the promoter/operator region may employ diverse mechanisms for transcriptional regulation of their genes.

Analysis of the Sequence Conservation of the Circadian Clock Protein KaiC

2013 ◽  
Vol 699 ◽  
pp. 184-188
Author(s):  
Liu Sen ◽  
Song Liu ◽  
Fei Yun Chen
Keyword(s):  
Circadian Rhythms ◽  
Circadian Clock ◽  
Feedback Loop ◽  
Sequence Variation ◽  
Protein Sequences ◽  
Circadian System ◽  
Site Variation ◽  
Key Residues ◽  
Clock Protein

Regulation of daily physiological functions with approximate a 24-hour periodicity, or circadian rhythms, is a characteristic of eukaryotes. So far, cyanobacteria are only known prokaryotes reported to possess circadian rhythmicity. The circadian system in cyanobacteria comprises both a post-translational oscillator (PTO) and a transcriptional/translational feedback loop (TTFL). The PTO can be reconstituted in vitro with three purified proteins (KaiA, KaiB, and KaiC) with the existence of ATP. Phase of the nanoclockwork has been associated with the phosphorylation states of KaiC, with KaiA promoting the phosphorylation of KaiC, and KaiB de-phosphorylating KaiC. Here we studied the sequence variation of 65 KaiC proteins in evolution, and determined some key residues in KaiC by analyzing the site variation rates of the protein sequences. These key residues could be used to study the key interactions of KaiC with KaiA and KaiB.

Understanding the highly efficient catalysis of prokaryotic peptide deformylases by shedding light on the determinants specifying the low activity of the human counterpart

2014 ◽  
Vol 70 (2) ◽  
pp. 242-252 ◽  
Author(s):  
Sonia Fieulaine ◽  
Michel Desmadril ◽  
Thierry Meinnel ◽  
Carmela Giglione
Keyword(s):  
Sequence Similarity ◽  
Three Dimensional ◽  
Binding Pocket ◽  
Dimensional Structure ◽  
Structural Basis ◽  
Human Counterpart ◽  
Low Activity

Peptide deformylases (PDFs), which are essential and ubiquitous enzymes involved in the removal of theN-formyl group from nascent chains, are classified into four subtypes based on the structural and sequence similarity of specific conserved domains. All PDFs share a similar three-dimensional structure, are functionally interchangeablein vivoand display similar propertiesin vitro, indicating that their molecular mechanism has been conserved during evolution. The human mitochondrial PDF is the only exception as despite its conserved fold it reveals a unique substrate-binding pocket together with an unusual kinetic behaviour. Unlike human PDF, the closely related mitochondrial PDF1As from plants have catalytic efficiencies and enzymatic parameters that are similar to those of other classes of PDFs. Here, the aim was to identify the structural basis underlying the properties of human PDF compared with all other PDFs by focusing on plant mitochondrial PDF1A. The construction of a chimaera composed of plant PDF1A with the nonrandom substitutions found in a conserved motif of its human homologue converted it into an enzyme with properties similar to the human enzyme, indicating the crucial role of these positions. The crystal structure of this human-like plant PDF revealed that substitution of two residues leads to a reduction in the volume of the ligand-binding site together with the introduction of negative charges, unravelling the origin of the weak affinity of human PDF for its substrate. In addition, the substitution of the two residues of human PDF modifies the transition state of the reaction through alteration of the network of interactions between the catalytic residues and the substrate, leading to an overall reduced reaction rate.

scholarly journals Characterization of the proteasome from the extremely halophilic archaeonHaloarcula marismortui

Archaea ◽  
2002 ◽  
Vol 1 (1) ◽  
pp. 53-61 ◽  
Author(s):  
B. Franzetti ◽  
G. Schoehn ◽  
D. Garcia ◽  
R. W. H. Ruigrok ◽  
G. Zaccai
Keyword(s):  
Three Dimensional ◽  
Halophilic Archaea ◽  
20S Proteasome ◽  
Misfolded Protein ◽  
Haloarcula Marismortui ◽  
Low Salt ◽  
Dimensional Reconstruction ◽  
The Stability

A 20S proteasome, comprising two subunits α and β, was purified from the extreme halophilic archaeonHaloarcula marismortui, which grows only in saturated salt conditions. The three-dimensional reconstruction of theH. marismortuiproteasome (Hm proteasome), obtained from negatively stained electron micrographs, is virtually identical to the structure of a thermophilic proteasome filtered to the same resolution. The stability of the Hm proteasome was found to be less salt-dependent than that of other halophilic enzymes previously described. The proteolytic activity of the Hm proteasome was investigated using the malate dehydrogenase fromH. marismortui(HmMalDH) as a model substrate. The HmMalDH denatures when the salt concentration is decreased below 2 M. Under these conditions, the proteasome efficiently cleaves HmMalDH during its denaturation process, but the fully denatured HmMalDH is poorly degraded. These in vitro experiments show that, at low salt concentrations, the 20S proteasome from halophilic archaea eliminates a misfolded protein.

Chloroplast division protein ARC3 acts on FtsZ2 by preventing filament bundling and enhancing GTPase activity

2018 ◽  
Vol 475 (1) ◽  
pp. 99-115 ◽  
Author(s):  
Rahamthulla S. Shaik ◽  
Min Woo Sung ◽  
Stanislav Vitha ◽  
Andreas Holzenburg
Keyword(s):  
Sequence Similarity ◽  
Three Dimensional ◽  
Chloroplast Division ◽  
Particle Analysis ◽  
Temperature Sensitive ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Z Ring

Chloroplasts evolved from cyanobacterial endosymbiotic ancestors and their division is a complex process initiated by the assembly of cytoskeletal FtsZ (Filamentous temperature sensitive Z) proteins into a ring structure at the division site (Z-ring). The cyanobacterial Z-ring positioning system (MinCDE proteins) is also conserved in chloroplasts, except that MinC was lost and replaced by the eukaryotic ARC3 (accumulation and replication of chloroplasts). Both MinC and ARC3 act as negative regulators of FtsZ assembly, but ARC3 bears little sequence similarity with MinC. Here, light scattering assays, co-sedimentation, GTPase assay and transmission electron microscopy in conjunction with single-particle analysis have been used to elucidate the structure of ARC3 and its effect on its main target in chloroplast division, FtsZ2. Analysis of FtsZ2 in vitro assembly reactions in the presence and absence of GMPCPP showed that ARC3 promotes FtsZ2 debundling and disassembly of existing filaments in a concentration-dependent manner and requires GTP hydrolysis. Three-dimensional reconstruction of ARC3 revealed an almost circular molecule in which the FtsZ-binding N-terminus and the C-terminal PARC6 (paralog of ARC6)-binding MORN (Membrane Occupation and Recognition Nexus) domain are in close proximity and suggest a model for PARC6-enabled binding of ARC3 to FtsZ2. The latter is corroborated by in vivo data.

scholarly journals The Bordetella Bps Polysaccharide Is Critical for Biofilm Development in the Mouse Respiratory Tract

2007 ◽  
Vol 189 (22) ◽  
pp. 8270-8276 ◽  
Author(s):  
Gina Parise Sloan ◽  
Cheraton F. Love ◽  
Neelima Sukumar ◽  
Meenu Mishra ◽  
Rajendar Deora
Keyword(s):  
Whooping Cough ◽  
Three Dimensional ◽  
Respiratory Pathogens ◽  
Adolescents And Adults ◽  
The Stability ◽  
Inert Surfaces ◽  
Biofilm Development

ABSTRACT Bordetellae are respiratory pathogens that infect both humans and animals. Bordetella bronchiseptica establishes asymptomatic and long-term to life-long infections of animal nasopharynges. While the human pathogen Bordetella pertussis is the etiological agent of the acute disease whooping cough in infants and young children, it is now being increasingly isolated from the nasopharynges of vaccinated adolescents and adults who sometimes show milder symptoms, such as prolonged cough illness. Although it has been shown that Bordetella can form biofilms in vitro, nothing is known about its biofilm mode of existence in mammalian hosts. Using indirect immunofluorescence and scanning electron microscopy, we examined nasal tissues from mice infected with B. bronchiseptica. Our results demonstrate that a wild-type strain formed robust biofilms that were adherent to the nasal epithelium and displayed architectural attributes characteristic of a number of bacterial biofilms formed on inert surfaces. We have previously shown that the Bordetella Bps polysaccharide encoded by the bpsABCD locus is critical for the stability and maintenance of three-dimensional structures of biofilms. We show here that Bps is essential for the formation of efficient nasal biofilms and is required for the colonization of the nose. Our results document a biofilm lifestyle for Bordetella in mammalian respiratory tracts and highlight the essential role of the Bps polysaccharide in this process and in persistence of the nares.

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