An integrated approach unravels a crucial structural property for the function of the insect steroidogenic Halloween protein Noppera-bo

AbstractEcdysteroids are the principal insect steroid hormones essential for insect development and physiology. In the last 18 years, several enzymes responsible for ecdysteroid biosynthesis, encoded by Halloween genes, have been identified and well characterized, both genetically and biochemically. However, none of these proteins have yet been characterized at the tertiary structure level. Here, we report an integrated in silico, in vitro, and in vivo analyses of the Halloween glutathione S-transferase (GST) protein, Noppera-bo (Nobo). We determine crystal structures of Drosophila melanogaster Nobo (DmNobo) complexed with glutathione and 17β-estradiol, a DmNobo inhibitor. 17β-estradiol almost fully occupied the putative ligand-binding pocket, and a prominent hydrogen bond formed between Asp113 of DmNobo and 17β-estradiol. Asp113 is essential for inhibiting DmNobo enzymatic activity by 17β-estradiol, as 17β-estradiol does not inhibit and physically interacts less with the Asp113Ala DmNobo point mutant. Asp113 is highly conserved among Nobo proteins, but not among other GSTs, implying that Asp113 is important for endogenous Nobo function. Indeed, a homozygous nobo allele possessing the Asp113Ala point mutation exhibits embryonic lethality with undifferentiated cuticle structure, a phenocopy of complete loss-of-function nobo homozygotes. These results suggest that the nobo family of GST proteins has acquired a unique amino acid residue, which seems to be essential for binding an endogenous sterol substrate to regulate ecdysteroid biosynthesis. This is the first study to reveal the structural characteristics of insect steroidogenic Halloween proteins. This study also provides basic insight into applied entomology for developing a new type of insecticides that specifically inhibit ecdysteroid biosynthesis.Significance StatementInsect molting and metamorphosis are drastic and dynamic biological processes and, therefore, have fascinated many scientists. Ecdysteroids represent one class of insect hormones that are indispensable for inducing molting and metamorphosis. It is well known that proteins responsible for catalyzing ecdysteroid biosynthesis reactions are encoded by “Halloween” genes, most of which have names of ghosts and phantoms. However, no studies have focused on the structural properties of these biosynthetic proteins. In this study, we addressed this unsolved issue and successfully unraveled a structural property that is crucial for the function of the fruit fly Halloween protein, Noppera-bo (a Japanese faceless ghost). This is the first study to reveal the structural characteristics of an insect steroidogenic Halloween protein.

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An integrated approach to unravel a crucial structural property required for the function of the insect steroidogenic Halloween protein Noppera-bo

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.011463 ◽

2020 ◽

Vol 295 (20) ◽

pp. 7154-7167 ◽

Cited By ~ 1

Author(s):

Kotaro Koiwai ◽

Kazue Inaba ◽

Kana Morohashi ◽

Sora Enya ◽

Reina Arai ◽

...

Keyword(s):

Structural Characteristics ◽

Integrated Approach ◽

Binding Pocket ◽

Loss Of Function ◽

Tertiary Structures ◽

Ecdysteroid Biosynthesis ◽

17Β Estradiol ◽

Unique Amino Acid

Ecdysteroids are the principal steroid hormones essential for insect development and physiology. In the last 18 years, several enzymes responsible for ecdysteroid biosynthesis encoded by Halloween genes were identified and genetically and biochemically characterized. However, the tertiary structures of these proteins have not yet been characterized. Here, we report the results of an integrated series of in silico, in vitro, and in vivo analyses of the Halloween GST protein Noppera-bo (Nobo). We determined crystal structures of Drosophila melanogaster Nobo (DmNobo) complexed with GSH and 17β-estradiol, a DmNobo inhibitor. 17β-Estradiol almost fully occupied the putative ligand-binding pocket and a prominent hydrogen bond formed between 17β-estradiol and Asp-113 of DmNobo. We found that Asp-113 is essential for 17β-estradiol–mediated inhibition of DmNobo enzymatic activity, as 17β-estradiol did not inhibit and physically interacted less with the D113A DmNobo variant. Asp-113 is highly conserved among Nobo proteins, but not among other GSTs, implying that this residue is important for endogenous Nobo function. Indeed, a homozygous nobo allele with the D113A substitution exhibited embryonic lethality and an undifferentiated cuticle structure, a phenocopy of complete loss-of-function nobo homozygotes. These results suggest that the nobo family of GST proteins has acquired a unique amino acid residue that appears to be essential for binding an endogenous sterol substrate to regulate ecdysteroid biosynthesis. To the best of our knowledge, ours is the first study describing the structural characteristics of insect steroidogenic Halloween proteins. Our findings provide insights relevant for applied entomology to develop insecticides that specifically inhibit ecdysteroid biosynthesis.

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Structural integrity of β-sheet assembly

Biochemical Society Transactions ◽

10.1042/bst0370671 ◽

2009 ◽

Vol 37 (4) ◽

pp. 671-676 ◽

Cited By ~ 35

Author(s):

Karen E. Marshall ◽

Louise C. Serpell

Keyword(s):

Spider Silk ◽

Structural Integrity ◽

Tertiary Structure ◽

Structural Characteristics ◽

Sequence Similarity ◽

Underlying Structure ◽

Β Sheet ◽

Proteins And Peptides

The folding of a protein from a sequence of amino acids to a well-defined tertiary structure is one of the most studied and enigmatic events to take place in biological systems. Relatively recently, it has been established that some proteins and peptides are able to take on conformations other than their native fold to form long fibres known as amyloid. In vivo, these are associated with misfolding diseases, such as Alzheimer's disease, Type 2 diabetes and the amyloidoses. In vitro, peptide assembly leads to amyloid-like fibres that have high stability, resistance to degradation and high tensile strength. Remarkably, despite the lack of any obvious sequence similarity between these fibrillogenic proteins and peptides, all amyloid fibrils share common structural characteristics and their underlying structure is known as ‘cross-β’. Nature is rich in β-sheet protein assemblies such as spider silk and other ‘useful’ amyloids such as curli from Escherichia coli, where the strength of fibrils is fundamental to their function.

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Use of light microscopy in the qualitative and quantitative study of liquid crystalline order

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100121727 ◽

1992 ◽

Vol 50 (1) ◽

pp. 264-265

Author(s):

Christopher Viney

Keyword(s):

Light Microscopy ◽

Liquid Crystalline ◽

Structural Characteristics ◽

Molecular Order ◽

Liquid Crystalline Phases ◽

Convenient Technique ◽

Liquid Crystalline Materials ◽

Transparent Windows

Light microscopy is a convenient technique for characterizing molecular order in fluid liquid crystalline materials. Microstructures can usually be observed under the actual conditions that promote the formation of liquid crystalline phases, whether or not a solvent is required, and at temperatures that can range from the boiling point of nitrogen to 600°C. It is relatively easy to produce specimens that are sufficiently thin and flat, simply by confining a droplet between glass cover slides. Specimens do not need to be conducting, and they do not have to be maintained in a vacuum. Drybox or other controlled environmental conditions can be maintained in a sealed chamber equipped with transparent windows; some heating/ freezing stages can be used for this purpose. It is relatively easy to construct a modified stage so that the generation and relaxation of global molecular order can be observed while specimens are being sheared, simulating flow conditions that exist during processing. Also, light only rarely affects the chemical composition or molecular weight distribution of the sample. Because little or no processing is required after collecting the sample, one can be confident that biologically derived materials will reveal many of their in vivo structural characteristics, even though microscopy is performed in vitro.

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In Silico Study of Potential Cross-Kingdom Plant MicroRNA Based Regulation in Chronic Myeloid Leukemia

Current Pharmacogenomics and Personalized Medicine ◽

10.2174/1875692118666200106113610 ◽

2020 ◽

Vol 17 (2) ◽

pp. 125-132

Author(s):

Marjanu Hikmah Elias ◽

Noraziah Nordin ◽

Nazefah Abdul Hamid

Keyword(s):

Targeted Therapy ◽

In Silico ◽

Structure Prediction ◽

Tertiary Structure ◽

Target Prediction ◽

In Silico Analysis ◽

Microrna Target ◽

Good Target

Background: Chronic Myeloid Leukaemia (CML) is associated with the BCRABL1 gene, which plays a central role in the pathogenesis of CML. Thus, it is crucial to suppress the expression of BCR-ABL1 in the treatment of CML. MicroRNA is known to be a gene expression regulator and is thus a good candidate for molecularly targeted therapy for CML. Objective: This study aims to identify the microRNAs from edible plants targeting the 3’ Untranslated Region (3’UTR) of BCR-ABL1. Methods: In this in silico analysis, the sequence of 3’UTR of BCR-ABL1 was obtained from Ensembl Genome Browser. PsRNATarget Analysis Server and MicroRNA Target Prediction (miRTar) Server were used to identify miRNAs that have binding conformity with 3’UTR of BCR-ABL1. The MiRBase database was used to validate the species of plants expressing the miRNAs. The RNAfold web server and RNA COMPOSER were used for secondary and tertiary structure prediction, respectively. Results: In silico analyses revealed that cpa-miR8154, csi-miR3952, gma-miR4414-5p, mdm-miR482c, osa-miR1858a and osa-miR1858b show binding conformity with strong molecular interaction towards 3’UTR region of BCR-ABL1. However, only cpa-miR- 8154, osa-miR-1858a and osa-miR-1858b showed good target site accessibility. Conclusion: It is predicted that these microRNAs post-transcriptionally inhibit the BCRABL1 gene and thus could be a potential molecular targeted therapy for CML. However, further studies involving in vitro, in vivo and functional analyses need to be carried out to determine the ability of these miRNAs to form the basis for targeted therapy for CML.

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TRIM27 interacts with Iκbα to promote the growth of human renal cancer cells through regulating the NF-κB pathway

BMC Cancer ◽

10.1186/s12885-021-08562-5 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Chengwu Xiao ◽

Wei Zhang ◽

Meimian Hua ◽

Huan Chen ◽

Bin Yang ◽

...

Keyword(s):

Cell Lines ◽

Prognostic Indicator ◽

The Cancer Genome Atlas ◽

Mrna Levels ◽

Loss Of Function ◽

Protein Levels ◽

Kaplan Meier ◽

Cancer Genome Atlas

Abstract Background The tripartite motif (TRIM) family proteins exhibit oncogenic roles in various cancers. The roles of TRIM27, a member of the TRIM super family, in renal cell carcinoma (RCC) remained unexplored. In the current study, we aimed to investigate the clinical impact and roles of TRIM27 in the development of RCC. Methods The mRNA levels of TRIM27 and Kaplan–Meier survival of RCC were analyzed from The Cancer Genome Atlas database. Real-time PCR and Western blotting were used to measure the mRNA and protein levels of TRIM27 both in vivo and in vitro. siRNA and TRIM27 were exogenously overexpressed in RCC cell lines to manipulate TRIM27 expression. Results We discovered that TRIM27 was elevated in RCC patients, and the expression of TRIM27 was closely correlated with poor prognosis. The loss of function and gain of function results illustrated that TRIM27 promotes cell proliferation and inhibits apoptosis in RCC cell lines. Furthermore, TRIM27 expression was positively associated with NF-κB expression in patients with RCC. Blocking the activity of NF-κB attenuated the TRIM27-mediated enhancement of proliferation and inhibition of apoptosis. TRIM27 directly interacted with Iκbα, an inhibitor of NF-κB, to promote its ubiquitination, and the inhibitory effects of TRIM27 on Iκbα led to NF-κB activation. Conclusions Our results suggest that TRIM27 exhibits an oncogenic role in RCC by regulating NF-κB signaling. TRIM27 serves as a specific prognostic indicator for RCC, and strategies targeting the suppression of TRIM27 function may shed light on future therapeutic approaches.

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An integrated approach for assessing the in vitro and in vivo redox-related effects of nanomaterials

Environmental Research ◽

10.1016/j.envres.2021.111083 ◽

2021 ◽

Vol 197 ◽

pp. 111083

Author(s):

Periklis Vardakas ◽

Zoi Skaperda ◽

Fotios Tekos ◽

Aikaterini-Flora Trompeta ◽

Aristidis Tsatsakis ◽

...

Keyword(s):

Integrated Approach

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Therapeutic Potentials of Syzygium fruticosum Fruit (Seed) Reflected into an Array of Pharmacological Assays and Prospective Receptors-Mediated Pathways

Life ◽

10.3390/life11020155 ◽

2021 ◽

Vol 11 (2) ◽

pp. 155

Author(s):

Jannatul Nasma Rupa Moni ◽

Md. Adnan ◽

Abu Montakim Tareq ◽

Md. Imtiazul Kabir ◽

A.S.M. Ali Reza ◽

...

Keyword(s):

Bioactive Compounds ◽

Integrated Approach ◽

Gas Chromatography Mass Spectrometry ◽

Clot Lysis ◽

Lipinski’S Rule ◽

Lysis Activity ◽

In Silico Studies ◽

Immobility Time

Syzygium fruticosum (SF), a valuable Bangladeshi fruit, is considered an alternative therapeutic agent. Mainly, seeds are used as nutritional phytotherapy to ease physical and mental status by preventing chronic diseases. Here, we scrutinized the S. fruticosum seed’s fundamental importance in traditional medicine by following an integrated approach combining in vivo, in vitro, and in silico studies. The SF was fractionated with different solvents, and the ethyl acetate fraction of SF (EaF-SF) was further studied. Mice treated with EaF-SF (200 and 400 mg/kg) manifested anxiolysis evidenced by higher exploration in elevated plus maze and hole board tests. Similarly, a dose-dependent drop of immobility time in a forced swimming test ensured significant anti-depressant activity. Moreover, higher dose treatment exposed reduced exploratory behaviour resembling decreased movement and prolonged sleeping latency with a quick onset of sleep during the open field and thiopental-induced sleeping tests, respectively. In parallel, EaF-SF significantly (p < 0.001) and dose-dependently suppressed acetic acid and formalin-induced pain in mice. Also, a noteworthy anti-inflammatory activity and a substantial (p < 0.01) clot lysis activity (thrombolytic) was observed. Gas chromatography-mass spectrometry (GC–MS) analysis resulted in 49 bioactive compounds. Among them, 12 bioactive compounds with Lipinski’s rule and safety confirmation showed strong binding affinity (molecular docking) against the receptors of each model used. To conclude, the S. fruticosum seed is a prospective source of health-promoting effects that can be an excellent candidate for preventing degenerative diseases.

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β-elemene alleviates airway stenosis via the ILK/Akt pathway modulated by MIR143HG sponging miR-1275

Cellular & Molecular Biology Letters ◽

10.1186/s11658-021-00261-0 ◽

2021 ◽

Vol 26 (1) ◽

Author(s):

Guoying Zhang ◽

Cheng Xue ◽

Yiming Zeng

Keyword(s):

Cell Cycle ◽

Cell Viability ◽

Cell Model ◽

Protective Efficacy ◽

Rabbit Model ◽

Akt Pathway ◽

Loss Of Function ◽

Airway Stenosis

Abstract Background We have previously found that β-elemene could inhibit the viability of airway granulation fibroblasts and prevent airway hyperplastic stenosis. This study aimed to elucidate the underlying mechanism and protective efficacy of β-elemene in vitro and in vivo. Methods Microarray and bioinformatic analysis were used to identify altered pathways related to cell viability in a β-elemene-treated primary cell model and to construct a β-elemene-altered ceRNA network modulating the target pathway. Loss of function and gain of function approaches were performed to examine the role of the ceRNA axis in β-elemene's regulation of the target pathway and cell viability. Additionally, in a β-elemene-treated rabbit model of airway stenosis, endoscopic and histological examinations were used to evaluate its therapeutic efficacy and further verify its mechanism of action. Results The hyperactive ILK/Akt pathway and dysregulated LncRNA-MIR143HG, which acted as a miR-1275 ceRNA to modulate ILK expression, were suppressed in β-elemene-treated airway granulation fibroblasts; β-elemene suppressed the ILK/Akt pathway via the MIR143HG/miR-1275/ILK axis. Additionally, the cell cycle and apoptotic phenotypes of granulation fibroblasts were altered, consistent with ILK/Akt pathway activity. In vivo application of β-elemene attenuated airway granulation hyperplasia and alleviated scar stricture, and histological detections suggested that β-elemene's effects on the MIR143HG/miR-1275/ILK axis and ILK/Akt pathway were in line with in vitro findings. Conclusions MIR143HG and ILK may act as ceRNA to sponge miR-1275. The MIR143HG/miR-1275/ILK axis mediates β-elemene-induced cell cycle arrest and apoptosis of airway granulation fibroblasts by modulating the ILK/Akt pathway, thereby inhibiting airway granulation proliferation and ultimately alleviating airway stenosis.

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Recent Developments in Inonotus obliquus (Chaga mushroom) Polysaccharides: Isolation, Structural Characteristics, Biological Activities and Application

Polymers ◽

10.3390/polym13091441 ◽

2021 ◽

Vol 13 (9) ◽

pp. 1441

Author(s):

Yangpeng Lu ◽

Yanan Jia ◽

Zihan Xue ◽

Nannan Li ◽

Junyu Liu ◽

...

Keyword(s):

Biological Activities ◽

Structural Characteristics ◽

Potential Candidate ◽

Health Food ◽

Inonotus Obliquus ◽

Recent Developments ◽

Inonotus Obliquus Polysaccharide ◽

Future Work

Inonotus obliquus (Chaga mushroom) is a kind of medicine and health food widely used by folk in China, Russia, Korea, and some occidental countries. Among the extracts from Inonotus obliquus, Inonotus obliquus polysaccharide (IOPS) is supposed to be one of the major bioactive components in Inonotus obliquus, which possesses antitumor, antioxidant, anti-virus, hypoglycemic, and hypolipidemic activities. In this review, the current advancements on extraction, purification, structural characteristics, and biological activities of IOPS were summarized. This review can provide significant insight into the IOPS bioactivities as their in vitro and in vivo data were summarized, and some possible mechanisms were listed. Furthermore, applications of IOPS were reviewed and discussed; IOPS might be a potential candidate for the treatment of cancers and type 2 diabetes. Besides, new perspectives for the future work of IOPS were also proposed.

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