scholarly journals Biochemical and functional characterization of the actin-binding activity of the B subunit of yeast vacuolar H+-ATPase

2008 ◽  
Vol 211 (7) ◽  
pp. 1102-1108 ◽  
Author(s):  
J. Zuo ◽  
S. Vergara ◽  
S. Kohno ◽  
L. S. Holliday
Keyword(s):  
Functional Characterization ◽  
Binding Activity ◽  
Actin Binding ◽  
B Subunit

scholarly journals Functional Characterization of a Novel Promoter Element Required for an Innate Immune Response in Drosophila

2003 ◽  
Vol 23 (22) ◽  
pp. 8272-8281 ◽  
Author(s):  
Hanna Uvell ◽  
Ylva Engström
Keyword(s):  
Gene Expression ◽  
Antimicrobial Peptide ◽  
Functional Characterization ◽  
Binding Activity ◽  
Innate Immune ◽  
Site Directed Mutagenesis ◽  
Promoter Element ◽  
Peptide Gene ◽  
Inducible Genes

ABSTRACT Innate immune reactions are crucial processes of metazoans to protect the organism against overgrowth of faster replicating microorganisms. Drosophila melanogaster is a precious model for genetic and molecular studies of the innate immune system. In response to infection, the concerted action of a battery of antimicrobial peptides ensures efficient killing of the microbes. The induced gene expression relies on translocation of the Drosophila Rel transcription factors Relish, Dif, and Dorsal to the nucleus where they bind to κB-like motifs in the promoters of the inducible genes. We have identified another putative promoter element, called region 1 (R1), in a number of antimicrobial peptide genes. Site-directed mutagenesis of the R1 site diminished Cecropin A1 (CecA1) expression in transgenic Drosophila larvae and flies. Infection of flies induced a nuclear R1-binding activity that was unrelated to the κB-binding activity in the same extracts. Although the R1 motif was required for Rel protein-mediated CecA1 expression in cotransfection experiments, our data argue against it being a direct target for the Drosophila Rel proteins. We propose that the R1 and κB motifs are targets for distinct regulatory complexes that act in concert to promote high levels of antimicrobial peptide gene expression in response to infection.

scholarly journals Purification by affinity chromatography and immunological characterization of a 110kDa component of the chick oviduct progesterone receptor

1984 ◽  
Vol 217 (3) ◽  
pp. 685-692 ◽  
Author(s):  
J M Renoir ◽  
J Mester ◽  
T Buchou ◽  
M G Catelli ◽  
P Tuohimaa ◽  
...  
Keyword(s):  
Progesterone Receptor ◽  
Affinity Chromatography ◽  
Specific Activity ◽  
Sedimentation Coefficient ◽  
Binding Activity ◽  
B Subunit ◽  
Stokes Radius ◽  
A Subunit ◽  
Final Yield

A 110kDa component of the chick oviduct progesterone receptor (PR) has been purified to homogeneity according to electrophoretic criteria and specific activity (assuming one progestagen-binding site/110kDa). The procedure involved affinity chromatography of 0.3 M-KCl-prepared cytosol, followed by DEAE-Sephacel chromatography (elution at 0.2 M-KCl). The final yield was about 12% in terms of binding activity. Properties of the 110kDa component indicate that it is identical with the ‘B’ subunit described previously [Stokes radius approximately 6.1 nm; sedimentation coefficient, (S20, w) approximately 4S; frictional ratio approximately 1.77]. It reacted with the IgG-G3 polyclonal antibody, but not with BF4 monoclonal antibody raised against the 8S molybdate-stabilized chick oviduct PR and reacting with its 90kDa component. Another progesterone-binding component, corresponding to the ‘A’ subunit, also previously described, was eluted from the DEAE-Sephacel column at approximately 0.08 M-KCl, and contained a peptide of molecular mass approx. 75-80kDa, which had S20, w approximately 4S in a sucrose gradient. This component was also recognized by IgG-G3, but not by BF4; it was very unstable in terms of hormone-binding activity.

scholarly journals Diversification of Transcription Factor NF-κB in Protists

2021 ◽  
Author(s):  
Leah M. Williams ◽  
Sainetra Sridhar ◽  
Jason Samaroo ◽  
Ebubechi K. Adindu ◽  
Anvitha Addanki ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Functional Characterization ◽  
Binding Activity ◽  
Life Stages ◽  
Rna Seq ◽  
Dna Binding Activity ◽  
Inhibitory Domain

In this report, we investigate the evolution of transcription factor NF-κB by examining its structure, activity, and regulation in two protists using phylogenetic, cellular, and biochemical techniques. In Capsaspora owczarzaki (Co), we find that full-length NF-κB has an N-terminal DNA-binding domain and a C-terminal Ankyrin (ANK) repeat inhibitory domain, and its DNA-binding activity is more similar to metazoan NF-κB rather than Rel proteins. As with mammalian NF-κB proteins, removal of the ANK repeats is required for Co-NF-κB to enter the nucleus, bind DNA, and activate transcription. However, C-terminal processing of Co-NF-κB is not induced by co-expression of IKK in human cells. Exogenously expressed Co-NF-κB localizes to the nucleus in Co cells. NF-κB mRNA and DNA-binding levels differ across three life stages of Capsaspora, suggesting distinct roles for NF-κB in these life stages. RNA-seq and GO analyses identify possible gene targets and biological functions of Co-NF-κB. We also show that three NF-κB-like proteins from the choanoflagellate Acanthoeca spectabilis (As) all consist of primarily the N-terminal conserved Rel Homology domain sequences of NF-κB, and lack C-terminal ANK repeats. All three As-NF-κB proteins constitutively enter the nucleus of human and Co cells, but differ in their DNA-binding and transcriptional activation activities. Furthermore, all three As-NF-κB proteins can form heterodimers, indicating that NF-κB diversified into multi-subunit families at least two times during evolution. Overall, these results present the first functional characterization of NF-κB in a taxonomic kingdom other than Animalia and provide information about the evolution and diversification of this biologically important transcription factor.

Physicochemical and functional characterization of trastuzumab-dkst, a trastuzumab biosimilar

2021 ◽  
Author(s):  
Parag Goyal ◽  
Jyoti Iyer ◽  
Laxmi Adhikary ◽  
Bhavesh Vats ◽  
Pradeep Kabadi ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Binding Activity ◽  
Order Structure ◽  
Repeat Dose ◽  
Antibody Dependent Cellular Cytotoxicity ◽  
Charge Heterogeneity ◽  
Tertiary Structures ◽  
Cynomolgus Monkeys ◽  
Comparative Similarity

Aims: Preclinical comparative similarity studies of trastuzumab-dkst, a Herceptin® biosimilar, are reported. Materials & methods: Primary sequence and higher-order structure and pharmacological mechanisms of action were compared using multiple techniques. Pharmacokinetics and repeat-dose toxicity were assessed in cynomolgus monkeys. Results: Primary structures were identical; secondary and tertiary structures were highly similar. Non-significant differences were observed for charge heterogeneity. Twelve of 13 glycan species were highly similar, with slightly higher total mannose levels in trastuzumab-dkst. FcγR and FcRn binding activity was highly similar. Each drug equally inhibited HER2+ cell proliferation, demonstrating equivalent relative potency in mediating HER2+ cell cytolysis by antibody-dependent cellular cytotoxicity. Pharmacokinetic and toxicological profiles in cynomolgus monkeys were similar. Conclusion: Trastuzumab-dkst, US-licensed trastuzumab and EU-approved trastuzumab demonstrate high structural and functional similarity.

scholarly journals IQGAP1, a Rac- and Cdc42-binding Protein, Directly Binds and Cross-links Microfilaments

1997 ◽  
Vol 137 (7) ◽  
pp. 1555-1566 ◽  
Author(s):  
Anne-Marie Bashour ◽  
Aaron T. Fullerton ◽  
Matthew J. Hart ◽  
George S. Bloom
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Filaments ◽  
Immunofluorescence Microscopy ◽  
Binding Activity ◽  
Cytochalasin D ◽  
Actin Binding ◽  
Purification And Characterization ◽  
Cross Links ◽  
Underlying Mechanisms

Activated forms of the GTPases, Rac and Cdc42, are known to stimulate formation of microfilament-rich lamellipodia and filopodia, respectively, but the underlying mechanisms have remained obscure. We now report the purification and characterization of a protein, IQGAP1, which is likely to mediate effects of these GTPases on microfilaments. Native IQGAP1 purified from bovine adrenal comprises two ∼190-kD subunits per molecule plus substoichiometric calmodulin. Purified IQGAP1 bound directly to F-actin and cross-linked the actin filaments into irregular, interconnected bundles that exhibited gel-like properties. Exogenous calmodulin partially inhibited binding of IQGAP1 to F-actin, and was more effective in the absence, than in the presence of calcium. Immunofluorescence microscopy demonstrated cytochalasin D–sensitive colocalization of IQGAP1 with cortical microfilaments. These results, in conjunction with prior evidence that IQGAP1 binds directly to activated Rac and Cdc42, suggest that IQGAP1 serves as a direct molecular link between these GTPases and the actin cytoskeleton, and that the actin-binding activity of IQGAP1 is regulated by calmodulin.

scholarly journals Purification and Functional Characterization of the C-Terminal Domain of the β-Actin-Binding Protein AIM1 In Vitro

Molecules ◽  
2018 ◽  
Vol 23 (12) ◽  
pp. 3281 ◽  
Author(s):  
Fang Wu ◽  
Liangkai Cheng ◽  
Qi Yu ◽  
Lin Zhang ◽  
Hong Li ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Structural Model ◽  
Functional Characterization ◽  
Actin Binding ◽  
Migration And Invasion ◽  
Binding Modes ◽  
C Terminus ◽  
Biology Research

The protein absent in melanoma 1 (AIM1) is a member of the βγ-crystal lens superfamily that is associated with the development of multiple cancers. The binding of AIM1 to β-actin affects the migration and invasion of prostate cancer epithelial cells. The C-terminus of AIM1 is required for the β-actin interaction. However, the characteristics of AIM1 in vitro and the interaction mode between AIM1 and β-actin remain unknown. We describe novel methods to prepare pure recombinant AIM1 and identify possible binding modes between AIM1 and β-actin; we also obtain the crystal of the first two βγ-crystallin domains of AIM1 (g1g2) for future structural biology research. We first express and purify AIM1 after cloning the sequence into a modified pET-28a_psp expression vector. Next, we define the minimum unit formed by the βγ-crystallin domain repeats that bound to β-actin and perform its physiological function. Finally, we made the structural model of the AIM1 g1g2 that can be used to guide future biomedical investigations and prostate cancer research.

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