Novel Procedure To Investigate the Effect of Phosphorylation on Protein Complex Formation in Vitro and in Cells†

Biochemistry ◽  
2008 ◽  
Vol 47 (7) ◽  
pp. 2153-2161 ◽  
Author(s):  
Makoto Rembutsu ◽  
Marc P. M. Soutar ◽  
Lidy Van Aalten ◽  
Robert Gourlay ◽  
C. James Hastie ◽  
...  
Keyword(s):  
Complex Formation ◽  
Protein Complex ◽  
Protein Complex Formation ◽  
In Cells

BIOLOGIC AND BIOCHEMICAL EFFECTS OF ANTI-ANDROGENS ON RAT VENTRAL PROSTATE

1974 ◽  
Vol 75 (2) ◽  
pp. 385-397 ◽  
Author(s):  
Jack Geller ◽  
Kevin McCoy
Keyword(s):  
Complex Formation ◽  
Protein Complex ◽  
Ventral Prostate ◽  
Common Denominator ◽  
Biochemical Effects ◽  
Protein Complex Formation ◽  
Biologic Effects ◽  
Rat Prostate

ABSTRACT To determine whether the similarity of biologic effects of two antiandrogens, cyproterone acetate (Cyp A) and edogesterone (PH-218), could be related to one or more common biochemical effects, we have compared the effects of both drugs on 3H testosterone (3HT) entry into cells, binding to specific cytosol and nuclear androphiles, and conversion to dihydrotestosterone (DHT). In chronic in vivo studies, both Cyp A and PH-218 reduced rat prostate weights by approximately 50% and specific cytosol steroid-protein complex formation by approximately 60 %. At the same time, Cyp A decreased the formation of nuclear steroid-protein complex to 10% of control values, compared with 40% for PH-218. In addition, Cyp A, but not PH-218, significantly decreased total 3HT uptake by the prostate. Similar effects of Cyp A on 3HT uptake, binding, and metabolism were noted in acute in vivo and in vitro experiments. PH-218 effects on these same parameters were reduced in acute, compared to chronic, studies. Neither drug significantly affected the conversion of T to DHT. Despite quantitative differences between Cyp A and PH-218, these studies support the concept that the biochemical common denominator for the biologic effects of anti-androgens is inhibition of specific steroid-protein complex formation in both cytosol and nucleus.

scholarly journals An AU-rich element in the 3' untranslated region of the spinach chloroplast petD gene participates in sequence-specific RNA-protein complex formation.

1995 ◽  
Vol 15 (4) ◽  
pp. 2010-2018 ◽  
Author(s):  
Q Chen ◽  
C C Adams ◽  
L Usack ◽  
J Yang ◽  
R A Monde ◽  
...  
Keyword(s):  
Complex Formation ◽  
Protein Complex ◽  
Untranslated Region ◽  
Rna Binding ◽  
Rbcl Gene ◽  
Spinach Chloroplast ◽  
Protein Complex Formation ◽  
Sequence Elements ◽  
Chloroplast Mrna

In chloroplasts, the 3' untranslated regions of most mRNAs contain a stem-loop-forming inverted repeat (IR) sequence that is required for mRNA stability and correct 3'-end formation. The IR regions of several mRNAs are also known to bind chloroplast proteins, as judged from in vitro gel mobility shift and UV cross-linking assays, and these RNA-protein interactions may be involved in the regulation of chloroplast mRNA processing and/or stability. Here we describe in detail the RNA and protein components that are involved in 3' IR-containing RNA (3' IR-RNA)-protein complex formation for the spinach chloroplast petD gene, which encodes subunit IV of the cytochrome b6/f complex. We show that the complex contains 55-, 41-, and 29-kDa RNA-binding proteins (ribonucleoproteins [RNPs]). These proteins together protect a 90-nucleotide segment of RNA from RNase T1 digestion; this RNA contains the IR and downstream flanking sequences. Competition experiments using 3' IR-RNAs from the psbA or rbcL gene demonstrate that the RNPs have a strong specificity for the petD sequence. Site-directed mutagenesis was carried out to define the RNA sequence elements required for complex formation. These studies identified an 8-nucleotide AU-rich sequence downstream of the IR; mutations within this sequence had moderate to severe effects on RNA-protein complex formation. Although other similar sequences are present in the petD 3' untranslated region, only a single copy, which we have termed box II, appears to be essential for in vitro protein binding. In addition, the IR itself is necessary for optimal complex formation. These two sequence elements together with an RNP complex may direct correct 3'-end processing and/or influence the stability of petD mRNA in chloroplasts.

scholarly journals Mutational Analysis of the Rhizobium etli recA Operator

1998 ◽  
Vol 180 (23) ◽  
pp. 6325-6331 ◽  
Author(s):  
Angels Tapias ◽  
Jordi Barbé
Keyword(s):  
Complex Formation ◽  
Protein Complex ◽  
Mutational Analysis ◽  
Rhizobium Meliloti ◽  
Regulatory Sequence ◽  
Rhizobium Etli ◽  
Wild Type ◽  
Protein Complex Formation

ABSTRACT Based upon our earlier studies (A. Tapias, A. R. Fernández de Henestrosa, and J. Barbé, J. Bacteriol. 179:1573–1579, 1997) we hypothesized that the regulatory sequence of the Rhizobium etli recA gene was TTGN11CAA. However, further detailed analysis of the R. etli recAoperator described in the present work suggests that it may in fact be GAACN7GTAC. This new conclusion is based upon PCR mutagenesis analysis carried out in the R. etli recAoperator, which indicates that the GAAC and GTAC submotifs found in the sequence GAACN7GTAC are required for the maximal stimulation of in vivo transcription and in vitro DNA-protein complex formation. This DNA-protein complex is also detected when the GAACN7GTAC wild-type sequence is modified to obtain GAACN7GAAC, GTACN7GTAC, or GAACN7GTTC. The wild-type promoters of the Rhizobium meliloti and Agrobacterium tumefaciens recA genes, which also contain the GAACN7GTAC sequence, compete with the R. etli recA promoter for the DNA-protein complex formation but not with mutant derivatives in any of these motifs, indicating that the R. etli, R. meliloti, andA. tumefaciens recA genes present the same regulatory sequence.

scholarly journals Analysis of Protein Complex Formation on Membrane Surfaces by Single Molecule Diffusion

2012 ◽  
Vol 102 (3) ◽  
pp. 183a
Author(s):  
Brian P. Ziemba ◽  
Jefferson D. Knight ◽  
Joseph J. Falke
Keyword(s):  
Complex Formation ◽  
Single Molecule ◽  
Protein Complex ◽  
Membrane Surfaces ◽  
Protein Complex Formation

scholarly journals Targeting WNT10B-Driven Signalling through Induction of FZD6 By Porcupine Inhibition in T Cell Acute Lymphoblastic Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3956-3956
Author(s):  
Adriana Cassaro ◽  
Francesca Lazzaroni ◽  
Giovanni Grillo ◽  
Gianluigi Reda ◽  
Roberto Cairoli ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Complex Formation ◽  
Wnt Signaling ◽  
Protein Complex ◽  
Lymphoblastic Leukemia ◽  
Protein Complex Formation ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Short Hairpin ◽  
Signaling Activation

Background Wnt/Fzd signaling is known to play a pervasive influence in hematopoietic stem cell maintenance, T-cell development in the thymus and function as well as an important role in T-cell acute lymphoblastic leukemia (T-ALL) establishment. We have previously described a recurrent rearrangement involving the WNT10Blocus (WNT10BR) expressing a transcript variant (WNT10BIVS1) in acute myeloid leukemia. To determine the occurrence of this rearrangement in T-ALL we analyzed retrospectively an italian cohort of patients (n=20) and detected the WNT10BRrearrangement with a high prevalence (14/20). We also confirmed the relevance of these findings to human disease, detecting the molecular circuit triggered by the WNT10B over-expression using the MOLT-4 T-ALL cell model.In this report, we examined the expression of components of the Wnt signaling cascade mediated by WNT10B and the effects of specific gene silencing by short hairpin RNA (shRNA) and exposure to the potent PORCN inhibitor (LGK974), or the TGFbRI inhibitor (A83-01) on the WNT10B-mediated Wnt signaling activation. Methods We used the T-ALL model MOLT-4 cell line to assess the WNT10B/FZD signaling axis driven by WNT10BR. In order to identify interaction between WNT10B and FZD receptors we performed in situ proximity ligation assay (PLA) a method used to visualize protein-protein interactions.MOLT4 cells were infected with WNT10B/WNT10BIVS1-shRNA silencing lentiviral vectors versus empty vector control and treated with increased concentration of LGK974 or A83-01, subsequently the effects of pharmacological inhibition on the WNT10B/FZD interactions and on Wnt effector proteins were evaluated by PLA and expression analyses. Cell proliferation and cell death were measured by EdU assay and Annexin-V/Propidium Iodide (PI) analyses. Results We found that WNT10BRdrives Wnt signaling activity in T-ALL through interaction of WNT10B with FZD6 receptor. The effects of WNT10B/FZD6 interaction on Wnt-mediated signal in MOLT-4 were interfered by short hairpin RNAs (shRNAs)-mediated gene silencing and by small molecules-mediated disruption of Wnt-dependent signaling. We performed WNT10BIVS1knockdown or pharmacological inhibition of WNT10B release by the porcupine (PORCN) inhibitor LGK974 and these in turn progressively down-modulate WNT10B/FZD6 protein complex formation and significantly impairs intracellular effectors and leukemic expansion. Finally, we induced interference to the WNT10B/FZD6 protein complex formation by exposure to the TGFbRI inhibitor A83-01 via inhibiting FZD6 expression, confirming its role in the WNT10B-mediated signaling activation. Conclusion Our study describes the molecular circuit of WNT10BR-mediated activation and highlight a strategy for a major improvement in T-ALL treatment.By altering FZD6-WNT10B complex formation, may provide the basis for therapeutic strategies to eradicate leukemic stem cells in patients selectively deployed depending on the underlying genetics of disease. Disclosures No relevant conflicts of interest to declare.

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