scholarly journals Involvement of Importin-4 in the Transport of Transition Protein 2 into the Spermatid Nucleus

2007 ◽  
Vol 28 (13) ◽  
pp. 4331-4341 ◽  
Author(s):  
M. M. Pradeepa ◽  
S. Manjunatha ◽  
V. Sathish ◽  
Shipra Agrawal ◽  
M. R. S. Rao
Keyword(s):  
Germ Cells ◽  
Nuclear Translocation ◽  
Binding Pocket ◽  
Pcr Analysis ◽  
Physical Interaction ◽  
Three Dimensional Model ◽  
Docking Simulations ◽  
Spermatid Nucleus ◽  
Transition Protein

ABSTRACT Mammalian spermiogenesis is characterized by a unique chromatin-remodeling process in which histones are replaced by transition protein 1 (TP1), TP2, and TP4, which are further replaced by protamines. We showed previously that the import of TP2 into the haploid spermatid nucleus requires the components of cytosol and ATP. We have now carried out a detailed analysis to characterize the molecular components underlying the nuclear translocation of TP2. Real-time PCR analysis of the expression of different importins in testicular germ cells revealed that importin-4 and importin-β3 are significantly up-regulated in tetraploid and haploid germ cells. We carried out physical interaction studies as well as an in vitro nuclear transport assay using recombinant TP2 and the nuclear localization signal of TP2 (TP2NLS) fused to glutathione S-transferase in digitonin-permeabilized, haploid, round spermatids and identified importin-4 to be involved in the import of TP2. A three-dimensional model of the importin-4 protein was generated using the crystal structure of importin-β1 as the template. Molecular docking simulations of TP2NLS with the importin-4 structure led to the identification of a TP2NLS binding pocket spanning the three helices (helices 21 to 23) of importin-4, which was experimentally confirmed by in vitro interaction and import studies with different deletion mutants of importin-4. In contrast to TP2, TP1 import was accomplished through a passive diffusion process.

scholarly journals Vascular endothelial growth factor regulates germ cell survival during establishment of spermatogenesis in the bovine testis

Reproduction ◽  
2009 ◽  
Vol 138 (4) ◽  
pp. 667-677 ◽  
Author(s):  
Kyle C Caires ◽  
Jeanene de Avila ◽  
Derek J McLean
Keyword(s):  
Vascular Endothelial Growth Factor ◽  
Growth Factor ◽  
Germ Cell ◽  
Cell Survival ◽  
Germ Cells ◽  
Pcr Analysis ◽  
Vascular Endothelial ◽  
Endothelial Growth Factor ◽  
Testis Tissue

Vascular endothelial growth factor-A (VEGFA) is a hypoxia-inducible peptide essential for angiogenesis and targets nonvascular cells in a variety of tissues and cell types. The objective of the current study was to determine the function of VEGF during testis development in bulls. We used an explant tissue culture and treatment approach to test the hypothesis that VEGFA-164 could regulate the biological activity of bovine germ cells. We demonstrate that VEGFA, KDR, and FLT1 proteins are expressed in germ and somatic cells in the bovine testis. Treatment of bovine testis tissue with VEGFA in vitro resulted in significantly more germ cells following 5 days of culture when compared with controls. Quantitative real-time RT-PCR analysis determined that VEGF treatment stimulated an intracellular response that prevents germ cell death in bovine testis tissue explants, as indicated by increased expression of BCL2 relative to BAX and decreased expression of BNIP3 at 3, 6, and 24 h during culture. Blocking VEGF activity in vitro using antisera against KDR and VEGF significantly reduced the number of germ cells in VEGF-treated testis tissue to control levels at 120 h. Testis grafting provided in vivo evidence that bovine testis tissue treated with VEGFA for 5 days in culture contained significantly more differentiating germ cells compared with controls. These findings support the conclusion that VEGF supports germ cell survival and sperm production in bulls.

scholarly journals Physical and functional interaction between the ID1 and p65 for activation of NF-κB

2012 ◽  
Vol 303 (3) ◽  
pp. C267-C277 ◽  
Author(s):  
Xiao Peng ◽  
Yuna Wang ◽  
Swapna Kolli ◽  
Junpeng Deng ◽  
Li Li ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Transcriptional Activation ◽  
Nuclear Translocation ◽  
Luciferase Assay ◽  
Physical Interaction ◽  
Site Mutation ◽  
Transcriptional Activation Domain ◽  
Apoptosis Protein

Inhibitor of differentiation or DNA binding-1 (ID1) is an important helix-loop-helix (HLH) transcription factor involved in diverse biological functions including cell differentiation, proliferation, apoptosis, and senescence. Recently, it was reported that ID1 can activate the NF-κB signaling pathway in a variety of cancer cells and a T cell line, but the mechanisms involved in ID1-mediated transactivation of NF-κB are not clear. In this study, we demonstrate by both in vitro pull-down assays and a cell-based in vivo two-hybrid system that ID1-mediated NF-κB activation is due to its physical interaction with p65. We have identified that the transcriptional activation domain (TAD) in p65 and the HLH domain in ID1 are vital for their interaction. Interestingly, a single site mutation (Leu76) in the HLH domain of ID1 protein drastically decreased its ability to bind with p65. Using a dual-luciferase assay, we demonstrated that the interaction between ID1 and p65 modulates activation of the NF-κB signaling pathway in vivo. In addition, we demonstrated that, by affecting the nuclear translocation of p65, ID1 is essential in regulating TNF-α-induced p65 recruitment to its downstream target, the cellular inhibitor of apoptosis protein 2 (cIAP2) promoter.

4-Coumarate:coenzyme A ligase isoform 3 from Piper nigrum (Pn4CL3) catalyzes the CoA thioester formation of 3,4-methylenedioxycinnamic and piperic acids

2020 ◽  
Vol 477 (1) ◽  
pp. 61-74
Author(s):  
Zhehao Jin ◽  
Juraithip Wungsintaweekul ◽  
Sang-Hoon Kim ◽  
Jeong-Han Kim ◽  
Yongho Shin ◽  
...  
Keyword(s):  
Catalytic Efficiency ◽  
Transcript Level ◽  
Binding Pocket ◽  
Phenylpropanoid Pathway ◽  
Black Pepper ◽  
Piper Nigrum ◽  
Pcr Analysis ◽  
Coumarate:Coa Ligase ◽  
Ferulic Acids

Black pepper, dried green fruit of Piper nigrum L., is a household spice most popular in the world. Piperine, the pungency compound of black pepper, is proposed to partially arise from phenylpropanoid pathway. In the biosynthesis of piperine, 4-coumarate:CoA ligase (4CLs) must play a pivotal role in activating intermediate acids to corresponding CoA thioesters to serve as substrates. Based on transcriptome data, we isolated three P. nigrum 4CL isoforms (Pn4CL1, -2, and -3) from unripe peppercorn. These Pn4CLs were expressed in E. coli for in vitro enzyme assay with putative substrates, namely cinnamic, coumaric, ferulic, piperonylic, 3,4-methylenedioxycinnamic (3,4-MDCA), and piperic acids. Phylogenetic analysis and substrate usage study indicated that Pn4CL1, active towards coumaric and ferulic acids, belongs to class I 4CL for lignin synthesis. Pn4CL2 was a typical cinnamate-specific coumarate:CoA ligase-like (CLL) protein. The Pn4CL3, as class II enzyme, exhibited general 4CL activity towards coumaric and ferulic acids. However, Pn4CL3 was also active towards piperonylic acid, 3,4-MDCA, and piperic acid. Pn4CL3 possessed ∼2.6 times higher catalytic efficiency (kcat/KM) towards 3,4-MDCA and piperic acid than towards coumaric and ferulic acids, suggesting its specific role in piperine biosynthesis. Different substrate preference among the Pn4CL isoforms can be explained by 3-dimensional protein structure modeling, which demonstrated natural variants in amino acid residues of binding pocket to accommodate different substrates. Quantitative PCR analysis of these isoforms indicated that Pn4CL1 transcript level was highest in the roots whereas Pn4CL2 in the fruits and Pn4CL3 in the leaves.

scholarly journals Blockade of Fgfr1 with PD166866 Protects Cartilage from the Catabolic Effects Induced by Interleukin-1β: A Genome-Wide Expression Profiles Analysis

Cartilage ◽  
2020 ◽  
pp. 194760352096820
Author(s):  
Lingxian Yi ◽  
Guihua Lan ◽  
Yue Ju ◽  
Xiushan Yin ◽  
Peipei Zhang ◽  
...  
Keyword(s):  
Nuclear Translocation ◽  
Expression Profiles ◽  
Gene Expression Pattern ◽  
Cartilage Explants ◽  
Interleukin 1Β ◽  
Receptor Interaction ◽  
Pcr Analysis ◽  
Therapeutic Effects ◽  
A Genome

Objective Previously we showed that genetic deletion of Fgfr1 in chondrocytes protected mice from progression of osteoarthritis (OA). The aim of this study is to evaluate the effect of PD166866, a potent selective inhibitor of Fgfr1, on cartilage degeneration induced by interleukin-1β (IL-1β) and to clarify underlying global gene expression pattern. Design Cartilage explants and primary rat chondrocytes were stimulated with IL-1β to establish an inflammatory OA in vitro model. The effects of PD166866 were determined by measuring the release of glycosaminoglycans (GAG) in cartilage explants and primary rat chondrocytes, and the underlying molecular mechanism was analyzed by microarray and RT-PCR analysis in primary chondrocytes. Results In cartilage explants, PD166866 significantly counteracts IL-β stimulated GAG release. In addition, PD166866 impede IL-1β-stimulated nuclear translocation of p65 in rat chondrocytes. Based on microarray analysis, a total of 67 and 132 genes with more than 1.5-fold changes were identified in IL-1β-treated versus control and PD166866 cotreatment versus IL-1β treatment alone, respectively. Only 19 thereof were coregulated by IL-1β and PD166866 simultaneously. GO and KEGG pathway analysis showed that some pathways, including “cytokine–cytokine receptor interaction,” “chemokine signaling pathway,” and “complement and coagulation cascades,” as well as some key genes like chemokines, complement, and matrix metalloproteinases may relevant for therapeutic application of Fgfr1 blockade in IL-1β-stimulated chondrocytes. Conclusion Our results clearly demonstrated that blockade of Fgfr1 with PD166866 could effectively suppress the catabolic effects induced by IL-1β, and elucidated whole genomic targets of Fgfr1 inhibition responsible for the therapeutic effects of Fgfr1 blockade against inflammatory OA.

scholarly journals A novel benzoxaborole is active against Escherichia coli and binds to FabI

2021 ◽  
Author(s):  
Soma Mandal ◽  
Tanya Parish
Keyword(s):  
Escherichia Coli ◽  
Acyl Carrier Protein ◽  
Target Identification ◽  
Binding Pocket ◽  
Physical Interaction ◽  
Gram Negative ◽  
E Coli ◽  
Drug Target Identification ◽  
Mycobacterial Growth

To combat the looming crisis of antimicrobial-resistant infections, there is an urgent need for novel antimicrobial discovery and drug target identification. The benzoxaborole series was previously identified as an inhibitor of mycobacterial growth. Here, we demonstrate that a benzoxaborole is also active against the Gram-negative bacterium Escherichia coli in vitro. We isolated resistant mutants of E. coli and subjected them to whole genome sequencing. We found mutations in the enoyl acyl carrier protein FabI. Mutations mapped around the active center site located close to the co-factor binding site. This site partially overlaps with the binding pocket of triclosan, a known FabI inhibitor. Similar to triclosan, the physical interaction of the benzoxaborole with FabI was dependent on the co-factor NAD + . Identification of the putative target of this compound in E. coli provides scope for further development and optimization of this series for Gram-negative pathogens.

scholarly journals Artesunate interacts with Vitamin D receptor to reverse mouse model of sepsis-induced immunosuppression via enhancing autophagy

2020 ◽  
Author(s):  
Shenglan Shang ◽  
Jiaqi Wu ◽  
Xiaoli Li ◽  
Xin Liu ◽  
Pan Li ◽  
...  
Keyword(s):  
Vitamin D ◽  
Inflammatory Cytokines ◽  
Vitamin D Receptor ◽  
Target Genes ◽  
Nuclear Translocation ◽  
Cecal Ligation ◽  
Physical Interaction ◽  
Pro Inflammatory Cytokines

AbstractBackground and PurposeImmunosuppression is the predominant cause of mortality for sepsis due to failure to eradicate invading pathogens. Unfortunately, no effective and specific drugs capable of reversing immunosuppression are available for clinical use. Increasing evidence implicates vitamin D receptor (VDR) involved in sepsis-induced immunosuppression. Herein, artesunate (AS) was discovered to reverse sepsis-induced immunosuppression and its molecular mechanism is investigated.Experimental ApproachEffect of artesunate on sepsis-induced immunosuppression was investigated in mice and in vitro. VDR was predicted to be an interacted candidate of AS by bioinformatics predict, then identified using PCR and immunoblotting. VDR, ATG16L1 and NF-κB p65 were modified to investigate the alteration of AS’s effect on pro-inflammatory cytokines release, bacteria clearance and autophagy activities in sepsis-induced immunosuppression.Key ResultsAS significantly reduced the mortality of cecal ligation and puncture (CLP)-induced sepsis immunosuppression mice challenged with Pseudomonas Aeruginosa, and enhanced proinflammatory cytokines release and bacterial clearance to reverse sepsis-induced immunosuppression in vivo and in vitro. Mechanically, AS interacted with VDR thereby inhibited the nuclear translocation of VDR, then influencing ATG16L1 transcription and subsequent autophagy activity. In addition, AS inhibited physical interaction between VDR and NF-κB p65 in LPS tolerance macrophages, then promoted nuclear translocation of NF-κB p65, which activated the transcription of NF-κB p65 target genes such as pro-inflammatory cytokines.Conclusion and ImplicationsOur findings provide an evidence that AS interacted with VDR to reverse sepsis-induced immunosuppression in an autophagy and NF-κB dependent way, highlighting a novel approach for sepsis treatment and drug repurposing of AS in the future.

scholarly journals Testis-specific transcriptional regulators selectively occupy BORIS-bound CTCF target regions in mouse male germ cells

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Samuel Rivero-Hinojosa ◽  
Sungyun Kang ◽  
Victor V. Lobanenkov ◽  
Gabriel E. Zentner
Keyword(s):  
Rna Polymerase Ii ◽  
Germ Cells ◽  
Transcriptional Regulators ◽  
Physical Interaction ◽  
Sequence Specificity ◽  
Male Gametes ◽  
Target Sites ◽  
Mature Spermatozoa

Abstract Despite sharing the same sequence specificity in vitro and in vivo, CCCTC-binding factor (CTCF) and its paralog brother of the regulator of imprinted sites (BORIS) are simultaneously expressed in germ cells. Recently, ChIP-seq analysis revealed two classes of CTCF/BORIS-bound regions: single CTCF target sites (1xCTSes) that are bound by CTCF alone (CTCF-only) or double CTCF target sites (2xCTSes) simultaneously bound by CTCF and BORIS (CTCF&BORIS) or BORIS alone (BORIS-only) in germ cells and in BORIS-positive somatic cancer cells. BORIS-bound regions (CTCF&BORIS and BORIS-only sites) are, on average, enriched for RNA polymerase II (RNAPII) binding and histone retention in mature spermatozoa relative to CTCF-only sites, but little else is known about them. We show that subsets of CTCF&BORIS and BORIS-only sites are occupied by several testis-specific transcriptional regulators (TSTRs) and associated with highly expressed germ cell-specific genes and histone retention in mature spermatozoa. We also demonstrate a physical interaction between BORIS and one of the analyzed TSTRs, TATA-binding protein (TBP)-associated factor 7-like (TAF7L). Our data suggest that CTCF and BORIS cooperate with additional TSTRs to regulate gene expression in developing male gametes and histone retention in mature spermatozoa, potentially priming certain regions of the genome for rapid activation following fertilization.

scholarly journals Identification and characterization of novel IGFBP5 interacting protein: evidence IGFBP5-IP is a potential regulator of osteoblast cell proliferation

2006 ◽  
Vol 290 (3) ◽  
pp. C900-C906 ◽  
Author(s):  
Yousef G. Amaar ◽  
Blanca Tapia ◽  
Shin-Tai Chen ◽  
David J. Baylink ◽  
Subburaman Mohan
Keyword(s):  
Cell Proliferation ◽  
Growth Factor ◽  
Nuclear Translocation ◽  
Bone Cells ◽  
Binding Protein ◽  
Pcr Analysis ◽  
Osteosarcoma Cell ◽  
Osteoblast Cell ◽  
Interacting Protein

Insulin-like growth factor binding protein-5 (IGFBP5) is a multifunctional protein, which acts not only as a traditional binding protein, but also functions as a growth factor independent of IGFs to stimulate bone formation. It has been predicted that the intrinsic growth factor action of IGFBP5 involves binding of IGFBP5 to a putative receptor to induce downstream signaling pathways and/or nuclear translocation of IGFBP5 to influence transcription of genes involved in osteoblast cell proliferation/differentiation. Our study indentified proteins that bound to IGFBP5 using IGFBP5 as bait in a yeast two-hybrid screen of the U2 human osteosarcoma cell cDNA library. One of the clones that interacted strongly with the bait under high-stringency conditions corresponded to a novel IGFBP5 interacting protein (IGFBP5-IP) encoded by a gene that resides in mouse chromosome 10. The interaction between IGFBP5-IP and IGFBP5 is confirmed by in vitro coimmunoprecipitation studies that used pFlag and IGFBP5 polyclonal antibody, and cell lysates overexpressing both IGFBP5-IP and IGFBP5. Northern blot and RT-PCR analysis showed that the IGFBP-IP is expressed in both untransformed normal human osteoblasts and in osteosarcoma cell lines, which are known to produce IGFBP5. To determine the roles of IGFBP5-IP, we evaluated the effect of blocking the expression of IGFBP5-IP on osteoblast proliferation. We found that using a IGFBP5-IP-specific small interfering-hairpin plasmid resulted in a decrease in both basal and IGFBP5-induced osteoblast cell proliferation. On the basis of these findings, we predict that IGFBP5-IP may act as intracellular mediator of growth promoting actions of IGFBP5 and perhaps other osteoregulatory agents in bone cells.

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