scholarly journals Defining the human C2H2 zinc finger degrome targeted by thalidomide analogs through CRBN

Science ◽  
2018 ◽  
Vol 362 (6414) ◽  
pp. eaat0572 ◽  
Author(s):  
Quinlan L. Sievers ◽  
Georg Petzold ◽  
Richard D. Bunker ◽  
Aline Renneville ◽  
Mikołaj Słabicki ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Small Molecules ◽  
Zinc Finger ◽  
Ubiquitin Ligase ◽  
Biochemical Analysis ◽  
Zinc Fingers ◽  
Functional Characterization ◽  
C2h2 Zinc Finger

The small molecules thalidomide, lenalidomide, and pomalidomide induce the ubiquitination and proteasomal degradation of the transcription factors Ikaros (IKZF1) and Aiolos (IKZF3) by recruiting a Cys2-His2 (C2H2) zinc finger domain to Cereblon (CRBN), the substrate receptor of the CRL4CRBN E3 ubiquitin ligase. We screened the human C2H2 zinc finger proteome for degradation in the presence of thalidomide analogs, identifying 11 zinc finger degrons. Structural and functional characterization of the C2H2 zinc finger degrons demonstrates how diverse zinc finger domains bind the permissive drug-CRBN interface. Computational zinc finger docking and biochemical analysis predict that more than 150 zinc fingers bind the drug-CRBN complex in vitro, and we show that selective zinc finger degradation can be achieved through compound modifications. Our results provide a rationale for therapeutically targeting transcription factors that were previously considered undruggable.

scholarly journals Structure, expression and in vitro functional characterization of a novel RNA binding zinc finger protein from Xenopus.

1991 ◽  
Vol 10 (10) ◽  
pp. 3087-3093 ◽  
Author(s):  
M. Köster ◽  
U. Kühn ◽  
T. Bouwmeester ◽  
W. Nietfeld ◽  
T. el-Baradi ◽  
...  
Keyword(s):  
Zinc Finger ◽  
Rna Binding ◽  
Zinc Finger Protein ◽  
Functional Characterization ◽  
Finger Protein

scholarly journals Characterization of Snail nuclear import pathways as representatives of C2H2 zinc finger transcription factors

2009 ◽  
Vol 122 (9) ◽  
pp. 1452-1460 ◽  
Author(s):  
J.-M. Mingot ◽  
S. Vega ◽  
B. Maestro ◽  
J. M. Sanz ◽  
M. A. Nieto
Keyword(s):  
Transcription Factors ◽  
Zinc Finger ◽  
Nuclear Import ◽  
C2h2 Zinc Finger

scholarly journals Mutations in the Second Linker of KLF1 Cause Congenital Non-Spherocytic Hemolytic Anemia Due to Global Reduction of In Vivo DNA-Binding Affinity

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1246-1246
Author(s):  
Stephen Huang ◽  
Kevin R. Gillinder ◽  
Annabel Sorolla ◽  
Emma Whitelaw ◽  
Andrew C Perkins
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Hemolytic Anemia ◽  
Zinc Finger ◽  
Fetal Liver ◽  
Zinc Fingers ◽  
Globin Gene ◽  
Flow Cytometric Analysis ◽  
Global Reduction

Abstract The mommeD45 mutation generates an amino acid transversion (H350R) within a conserved linker peptide between zinc fingers two and three of Klf1 (linker 2). Klf1H350R/H350R mice have mild compensated microcytic anaemia 1. Mice Carrying the H350R mutation were interbred with Klf1+/- mice. Klf1H350R/-mice have severe perinatal haemolytic anaemia, jaundice and marked splenomegaly. Haematological evaluation of these mice shows similar phenotypes to human patients who are compound heterozygotes for null and linker 2 mutations in KLF12. Analysis of Klf1H350R/- fetal liver by flow cytometry showed an increase in circulating immature CD71+ Ter119+ erythroblasts. In the bone marrow, a marked reduction in mature (Cd71- Ter119+) red blood cells was observed. Flow cytometric analysis of the spleen from Klf1H350R/- animals revealed an expansion of erythroid cells consistent with extramedullary erythropoiesis. ChIP-seq for Klf1 in 14.5DPC fetal liver from Klf1H350R/H350R mice revealed no loss in specificity when compared to wildtype Klf1, but a global reduction in affinity. Affinity measurements of recombinant zinc finger domains in vitro will be presented. By RNA-seq, we observed significantly lower expression of Klf1 target genes in mice homozygous for the H350R mutation compared to mice carrying a wildtype allele. And this correlates with reduced DNA binding observed in ChIP-seq and in vitro assays. Previous studies of the linkers in C2H2 zinc finger transcription factors have revealed their necessity as structural and regulatory components for the C2H2 class of transcription factors. Our results show the second linker of Klf1 plays an indirect role in DNA-binding and does not act just as a spacer for the zinc fingers. References: 1. Sorolla A, Tallack MR, Oey H, et al. Identification of novel hypomorphic and null mutations in Klf1 derived from a genetic screen for modifiers of alpha-globin transgene variegation. Genomics. 2015;105(2):116-122. 2. Viprakasit V, Ekwattanakit S, Riolueang S, et al. Mutations in Kruppel-like factor 1 cause transfusion-dependent hemolytic anemia and persistence of embryonic globin gene expression. Blood. 2014;123(10):1586-1595. Disclosures Perkins: Novartis Oncology: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria.

scholarly journals Functional identification of ygiP as a positive regulator of the ttdA-ttdB-ygjE operon

Microbiology ◽  
2006 ◽  
Vol 152 (7) ◽  
pp. 2129-2135 ◽  
Author(s):  
Taku Oshima ◽  
Francis Biville
Keyword(s):  
Functional Characterization ◽  
Anaerobic Growth ◽  
Functional Identification ◽  
New Members ◽  
E Coli ◽  
Inner Membrane Protein ◽  
Tartrate Dehydratase

Functional characterization of unknown genes is currently a major task in biology. The search for gene function involves a combination of various in silico, in vitro and in vivo approaches. Available knowledge from the study of more than 21 LysR-type regulators in Escherichia coli has facilitated the classification of new members of the family. From sequence similarities and its location on the E. coli chromosome, it is suggested that ygiP encodes a lysR regulator controlling the expression of a neighbouring operon; this operon encodes the two subunits of tartrate dehydratase (TtdA, TtdB) and YgiE, an integral inner-membrane protein possibly involved in tartrate uptake. Expression of tartrate dehydratase, which converts tartrate to oxaloacetate, is required for anaerobic growth on glycerol as carbon source in the presence of tartrate. Here, it has been demonstrated that disruption of ygiP, ttdA or ygjE abolishes tartrate-dependent anaerobic growth on glycerol. It has also been shown that tartrate-dependent induction of the ttdA-ttdB-ygjE operon requires a functional YgiP.

scholarly journals Functional Characterization of the mazEF Toxin-Antitoxin System in the Pathogenic Bacterium Agrobacterium tumefaciens

2021 ◽  
Vol 9 (5) ◽  
pp. 1107
Author(s):  
Wonho Choi ◽  
Yoshihiro Yamaguchi ◽  
Ji-Young Park ◽  
Sang-Hyun Park ◽  
Hyeok-Won Lee ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Physiological Function ◽  
Functional Characterization ◽  
Site Directed Mutagenesis ◽  
In Vitro Assays ◽  
Cell Growth Inhibition ◽  
The Right

Agrobacterium tumefaciens is a pathogen of various plants which transfers its own DNA (T-DNA) to the host plants. It is used for producing genetically modified plants with this ability. To control T-DNA transfer to the right place, toxin-antitoxin (TA) systems of A. tumefaciens were used to control the target site of transfer without any unintentional targeting. Here, we describe a toxin-antitoxin system, Atu0939 (mazE-at) and Atu0940 (mazF-at), in the chromosome of Agrobacterium tumefaciens. The toxin in the TA system has 33.3% identity and 45.5% similarity with MazF in Escherichia coli. The expression of MazF-at caused cell growth inhibition, while cells with MazF-at co-expressed with MazE-at grew normally. In vivo and in vitro assays revealed that MazF-at inhibited protein synthesis by decreasing the cellular mRNA stability. Moreover, the catalytic residue of MazF-at was determined to be the 24th glutamic acid using site-directed mutagenesis. From the results, we concluded that MazF-at is a type II toxin-antitoxin system and a ribosome-independent endoribonuclease. Here, we characterized a TA system in A. tumefaciens whose understanding might help to find its physiological function and to develop further applications.

Functional characterization of two bZIP transcription factors in Verticillium dahliae

Gene ◽  
2017 ◽  
Vol 626 ◽  
pp. 386-394 ◽  
Author(s):  
Yulin Fang ◽  
Dianguang Xiong ◽  
Longyan Tian ◽  
Chen Tang ◽  
Yonglin Wang ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Verticillium Dahliae ◽  
Functional Characterization ◽  
Bzip Transcription Factors

scholarly journals NFB-01. FUNCTIONAL CHARACTERIZATION OF ATRX LOSS IN NF1-ASSOCIATED GLIOMA AND MPNST

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii417-iii418
Author(s):  
Ming Yuan ◽  
Karlyne Reilly ◽  
Christine Pratilas ◽  
Christopher Heaphy ◽  
Fausto Rodriguez
Keyword(s):  
Cell Lines ◽  
Functional Characterization ◽  
Tert Promoter ◽  
Aggressive Tumors ◽  
Sirna Knockdown ◽  
Nih 3T3 ◽  
Vitro Effect ◽  
Murine Glioma

Abstract To identify the biologic relevance of ATRX loss in NF1-associated gliomagenesis, we studied the effects of Atrx loss using four previously characterized Nf1+/-Trp53+/- murine glioma lines. Lines 130G#3 and 158D#8 (corresponding to grade IV and III gliomas, respectively) displayed preserved ATRX protein expression compared to NIH-3T3 cells. We studied the effects of Atrx knockdown in these two lines in the presence and absence of the TERT inhibitor, BIRBR1532. Using a telomere-specific FISH assay, we identified increased signal intensity after Atrx knockdown, only in the presence of the TERT inhibitor. These features are reminiscent of ALT, although there were no significant alterations in cell growth. Next, we studied the effect of ATRX loss in MPNST lines ST88-14, NF90-8, STS-26T. These cell lines all expressed ATRX and DAXX. However, STS-26T contained a TERT promoter mutation and ST88-14 had a known SNP in the TERT promoter, while NF90-8 had no alterations. ATRX siRNA knockdown showed no significant effects in cell proliferation or apoptosis. However, ATRX knockdown resulted in rare ultra-bright foci, indicative of ALT. Next, we studied the in vitro effect of the ATR inhibitor VE-821 in MPNST cell lines. Only NF90-8 (lacking TERT alterations) demonstrated a decrease in growth after ATRX knockdown and VE-821 treatment. However, ATRX knockdown alone did not affect sensitivity to carboplatin. Our findings further support a role for ATRX loss with subsequent ALT activation in a biologic subset of NF1-associated malignancies, thereby opening an opportunity for therapeutic targeting of these aggressive tumors using specific classes of drugs.

Functional Characterization of GABAA Receptor Ligands In Vitro

2004 ◽  
pp. 85-94
Author(s):  
Bjarke Ebert ◽  
Sally Anne Thompson ◽  
Signe Í. Stórustovu ◽  
Keith A. Wafford
Keyword(s):  
Gabaa Receptor ◽  
Functional Characterization ◽  
Receptor Ligands

scholarly journals Biochemical and Functional Characterization of Anthocyanidin Reductase (ANR) from Mangifera indica L.

Molecules ◽  
2018 ◽  
Vol 23 (11) ◽  
pp. 2876 ◽  
Author(s):  
Lin Tan ◽  
Mei Wang ◽  
Youfa Kang ◽  
Farrukh Azeem ◽  
Zhaoxi Zhou ◽  
...  
Keyword(s):  
Enzyme Assay ◽  
Molecular Mechanisms ◽  
Mangifera Indica ◽  
Functional Characterization ◽  
Citrate Buffer ◽  
Anthocyanidin Reductase ◽  
High Amino Acid ◽  
Optimum Ph

Mango (Mangifera indica L.) is abundant in proanthocyanidins (PAs) that are important for human health and plant response to abiotic stresses. However, the molecular mechanisms involved in PA biosynthesis still need to be elucidated. Anthocyanidin reductase (ANR) catalyzes a key step in PA biosynthesis. In this study, three ANR cDNAs (MiANR1-1,1-2,1-3) were isolated from mango, and expressed in Escherichia coli. In vitro enzyme assay showed MiANR proteins convert cyanidin to their corresponding flavan-3-ols, such as (−)-catechin and (−)-epicatechin. Despite high amino acid similarity, the recombinant ANR proteins exhibited differences in enzyme kinetics and cosubstrate preference. MiANR1-2 and MiANR1-3 have the same optimum pH of 4.0 in citrate buffer, while the optimum pH for MiANR1-1 is pH 3.0 in phosphate buffer. MiANR1-1 does not use either NADPH or NADH as co-substrate while MiANR1-2/1-3 use only NADPH as co-substrate. MiANR1-2 has the highest Km and Vmax for cyanidin, followed by MiANR1-3 and MiANR1-1. The overexpression of MiANRs in ban mutant reconstructed the biosynthetic pathway of PAs in the seed coat. These data demonstrate MiANRs can form the ANR pathway, leading to the formation of two types of isomeric flavan-3-ols and PAs in mango.

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