Easy Expression and Purification of Fluorescent N-Terminal BCL11B CCHC Zinc Finger Domain

Susemihl, Anne; Nagel, Felix; Grabarczyk, Piotr; Schmidt, Christian A.; Delcea, Mihaela

doi:10.3390/molecules26247576

Cited by 4 publications

(3 citation statements)

References 46 publications

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“…Our detected TFs were classified into three groups including TF classes with 1–10 TFs such as β-sheet, lg-fold, other α-Helix classes; TF classes with 10–20 TFs such as winged helix–turn–helix, helix–turn-helix, zipper-type; and TF classes with more than 20 TFs such as zinc-coordinating. Zinc finger proteins, the most abundant and structurally diverse groups of protein in nature, act as TFs and function in different cellular processes, including cell proliferation [ 60 , 61 , 62 ]. B-cell lymphoma/leukemia 11B (BCL11B) is one member of the large family of zinc finger proteins [ 61 ].…”

Section: Discussionmentioning

confidence: 99%

“…Zinc finger proteins, the most abundant and structurally diverse groups of protein in nature, act as TFs and function in different cellular processes, including cell proliferation [ 60 , 61 , 62 ]. B-cell lymphoma/leukemia 11B (BCL11B) is one member of the large family of zinc finger proteins [ 61 ]. Its inhibition results in growth retardation and apoptosis in T-cells [ 63 ].…”

Section: Discussionmentioning

confidence: 99%

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Gene Networks and Pathways Involved in LPS-Induced Proliferative Response of Bovine Endometrial Epithelial Cells

Najafi

Guo

Andersson

et al. 2022

Genes

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Lipopolysaccharide (LPS) is a component of the outer membrane of Gram-negative bacteria involved in the pathogenic processes leading to mastitis and metritis in animals such as dairy cattle. LPS causes cell proliferation associated with endometrium inflammation. Former in vitro studies have demonstrated that LPS induces an intense stimulation of the proliferation of a pure population of bovine endometrial epithelial cells. In a follow-up transcriptomic study based on RNA-sequencing data obtained after 24 h exposure of primary bovine endometrial epithelial cells to 0, 2, and 8 μg/mL LPS, 752 and 727 differentially expressed genes (DEGs) were detected between the controls and LPS-treated samples that encode proteins known to be associated with either proliferation or apoptosis, respectively. The present bioinformatic analysis was performed to decipher the gene networks involved to obtain a deeper understanding of the mechanisms underlying the proliferative and apoptosis processes. Our findings have revealed 116 putative transcription factors (TFs) and the most significant number of interactions between these TFs and DEGs belong to NFKβ1, TP53, STAT1, and HIF1A. Moreover, our results provide novel insights into the early signaling and metabolic pathways in bovine endometrial epithelial cells associated with the innate immune response and cell proliferation to Escherichia coli-LPS infection. The results further indicated that LPS challenge elicited a strong transcriptomic response, leading to potent activation of pro-inflammatory pathways that are associated with a marked endometrial cancer, Toll-like receptor, NFKβ, AKT, apoptosis, and MAPK signaling pathways. This effect may provide a mechanistic explanation for the relationship between LPS and cell proliferation.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Gene Networks and Pathways Involved in LPS-Induced Proliferative Response of Bovine Endometrial Epithelial Cells

Najafi

Guo

Andersson

et al. 2022

Genes

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“…Protein purity and homogeneity were verified by tricine SDS PAGE and analytical size exclusion chromatography. Calibration of the analytical size exclusion can be found in the work of Susemihl et al 19 …”

Section: Methodsmentioning

confidence: 99%

Identification of Kazal Inhibitor Scaffolds with Identical Canonical Binding Loops and Their Effects on Binding Properties

et al. 2023

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Kazal inhibitors hold high potential as scaffolds for therapeutic molecules, taking advantage of the easily exchangeable canonical binding loop. Different Kazal inhibitor backbones have been suggested to be therapeutically useful, but the impact of different Kazallike scaffolds on binding properties is still largely unknown. Here, we identified trypsin-targeting human serine protease inhibitor Kazal type 1 (SPINK1) homologues in different mammalian species that cluster in two P2−P1 combinations, implying the coevolution of these residues. We generated loop exchange variants of human SPINK1 for comparison with Kazal inhibitors from related species. Using comprehensive biophysical characterization of the inhibitor−enzyme interactions, we found not only affinity but also pH resistance to be highly backbonedependent. Differences are mostly observed in complex stability, which varies by over one order of magnitude. We provide clear evidence for high backbone dependency within the Kazal family. Hence, when designing Kazal inhibitor-based therapeutic molecules, testing different backbones after optimizing the canonical binding loop can be beneficial and may result in increased affinity, complex stability, specificity, and pH resistance.

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Solution structure of the Z0 domain from transcription repressor BCL11A sheds light on the sequence properties of protein-binding zinc-fingers

Harris,

Whitehead,

Alexandrescu

2024

Preprint

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The transcription repressor BCL11A, which governs the switch from fetal to adult hemoglobin during development, is the target of the first FDA-approved CRISPR/Cas9 gene-editing therapy in humans. By targeting BCL11A, fetal hemoglobin expression is de-repressed to substitute for defective adult hemoglobin in inherited diseases including beta-thalassemia and sickle-cell anemia. BCL11A has six CCHH-type zinc-fingers of which domains 4-6 are necessary and sufficient for dsDNA binding. Here, we focus on the CCHC-type ZNF at the N-terminus of BCL11A (residues 46-72), Z0, thought to modulate oligomerization of the transcription repressor. Using NMR and CD spectroscopy, Z0 is shown to be a thermostable CCHC zinc-finger with a pM dissociation constant for zinc. The NMR structure of Z0 has a prototypical beta-beta-alpha fold, with a hydrophobic knob comprising about half the structure. The unusual proportion of hydrophobic residues in Z0 led us to investigate if this is more general in zinc-fingers that do not bind dsDNA. We used the ZF and WebLogo servers to examine sequences of zinc fingers with demonstrated DNA-binding function, non-binders, and the CCHC-type family of protein-binders. DNA-binders are distinguished by contiguous stretches of high-scoring zinc-fingers. Non-DNA-binders show a depletion of polar residues at the positions expected to contact nucleotides and increased divergence from sequence consensus making these domains more likely to be annotated as atypical, degenerate, or to be missed as zinc-fingers. We anticipate these sequence patterns will help distinguish DNA-binders from non-binders, an open problem in the functional understanding of zinc-finger motifs.

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Easy Expression and Purification of Fluorescent N-Terminal BCL11B CCHC Zinc Finger Domain

Cited by 4 publications

References 46 publications

Gene Networks and Pathways Involved in LPS-Induced Proliferative Response of Bovine Endometrial Epithelial Cells

Gene Networks and Pathways Involved in LPS-Induced Proliferative Response of Bovine Endometrial Epithelial Cells

Identification of Kazal Inhibitor Scaffolds with Identical Canonical Binding Loops and Their Effects on Binding Properties

Solution structure of the Z0 domain from transcription repressor BCL11A sheds light on the sequence properties of protein-binding zinc-fingers

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