Dynamic BTB-domain filaments promote clustering of ZBTB proteins.

The formation of dynamic protein filaments contributes to various biological functions by clustering individual molecules together and enhancing their binding to ligands. We report such a propensity for the BTB domains of certain proteins from the ZBTB family, a large eukaryotic transcription factor family implicated in differentiation and cancer. Working with Xenopus laevis and human proteins, we solved the crystal structures of filaments formed by dimers of the BTB domains of ZBTB8A and ZBTB18 and demonstrated concentration-dependent higher-order assemblies of these dimers in solution. In cells, the BTB-domain filamentation supports clustering of full-length human ZBTB8A and ZBTB18 into dynamic nuclear foci and contributes to the ZBTB18-mediated repression of a reporter gene. The BTB domains of up to 21 human ZBTB family members and two related proteins, NACC1 and NACC2, are predicted to behave in a similar manner. Our results suggest that filamentation is a more common feature of transcription factors than is currently appreciated.

Zinc Finger and BTB Domain-Containing 20: A Newly Emerging Player in Pathogenesis and Development of Human Cancers.

Zinc finger and BTB domain-containing 20 (ZBTB20), which was initially identified in human dendritic cells, belongs to a family of transcription factors (TFs) with an N-terminal BTB domain and one or more C-terminal DNA-binding zinc finger domains. Under physiological conditions, ZBTB20 acts as a transcriptional repressor in cellular development and differentiation, metabolism, and innate immunity. Interestingly, multiple lines of evidence from mice and human systems have revealed the importance of ZBTB20 in the pathogenesis and development of cancers. ZBTB20 is not only a hotspot of genetic variation or fusion in many types of human cancers, but also a key TF or intermediator involving in the dysregulation of cancer cells. Given the diverse functions of ZBTB20 in both health and disease, we herein summarize the structure and physiological roles of ZBTB20, with an emphasis on the latest findings on tumorigenesis and cancer progression.

BTB domain mutations perturbing KCTD15 oligomerisation cause a distinctive frontonasal dysplasia syndrome.

INTRODUCTION: KCTD15 encodes an oligomeric BTB domain protein reported to inhibit neural crest formation through repression of Wnt/beta-catenin signalling, as well as transactivation by TFAP2. Heterozygous missense variants in the closely related paralogue KCTD1 cause scalp-ear-nipple syndrome. METHODS: Exome sequencing was performed on a two-generation family affected by a distinctive phenotype comprising a lipomatous frontonasal malformation, anosmia, cutis aplasia of the scalp and/or sparse hair, and congenital heart disease. Identification of a de novo missense substitution within KCTD15 led to targeted sequencing of DNA from a similarly affected sporadic patient, revealing a different missense mutation. Structural and biophysical analyses were performed to assess the effects of both amino acid substitutions on the KCTD15 protein. RESULTS: A heterozygous c.310G>C variant encoding p.(Asp104His) within the BTB domain of KCTD15 was identified in an affected father and daughter and segregated with the phenotype. In the sporadically affected patient, a de novo heterozygous c.263G>A variant encoding p.(Gly88Asp) was present in KCTD15. Both substitutions were found to perturb the pentameric assembly of the BTB domain. A crystal structure of the BTB domain variant p.(Gly88Asp) revealed a closed hexameric assembly, whereas biophysical analyses showed that the p.(Asp104His) substitution resulted in a monomeric BTB domain likely to be partially unfolded at physiological temperatures. CONCLUSION: BTB domain substitutions in KCTD1 and KCTD15 cause clinically overlapping phenotypes involving craniofacial abnormalities and cutis aplasia. The structural analyses demonstrate that missense substitutions act through a dominant negative mechanism by disrupting the higher order structure of the KCTD15 protein complex.

Hepatic Zbtb18 (Zinc Finger and BTB Domain Containing 18) alleviates hepatic steatohepatitis via FXR (Farnesoid X Receptor).

A lasting imbalance between fatty acid synthesis and consumption leads to non-alcoholic fatty liver disease (NAFLD), coupled with hepatitis and insulin resistance. Yet the details of the underlying mechanisms are not fully understood. Here, we unraveled that the expression of the transcription factor Zbtb18 is markedly decreased in the livers of both patients and murine models of NAFLD. Hepatic Zbtb18 knockout promoted NAFLD features like impaired energy expenditure and fatty acid oxidation (FAO), and induced insulin resistance. Conversely, hepatic Zbtb18 overexpression alleviated hepato-steatosis, insulin resistance, and hyperglycemia in mice fed on a high-fat diet (HFD) or in diabetic mice. Notably, in vitro and in vivo mechanistic studies revealed that Zbtb18 transcriptional activation of Farnesoid X receptor (FXR) mediated FAO and Clathrin Heavy Chain (CLTC) protein hinders NLRP3 inflammasome activity. This key mechanism by which hepatocyte's Zbtb18 expression alleviates NAFLD and consequent liver fibrosis was further verified by FXR's deletion and forced expression in mice and cultured mouse primary hepatocytes (MPHs). Moreover, CLTC deletion significantly abrogated the hepatic Zbtb18 overexpression-driven inhibition of NLRP3 inflammasome activity in macrophages. Altogether, Zbtb18 transcriptionally activates the FXR-mediated FAO and CLTC expression, which inhibits NLRP3 inflammasome's activity alleviating inflammatory stress and insulin resistance, representing an attractive remedy for hepatic steatosis and fibrosis.

Formononetin enhances the chemosensitivity of triple negative breast cancer via BTB domain and CNC homolog 1-mediated mitophagy pathways.

This study aimed to investigate the effects of formononetin on triple negative breast cancer (TNBC). Clinical samples were collected from patients with TNBC. Overall survival rates were evaluated using the Kaplan-Meier method. Gene expression was determined using immunohistochemistry, immunofluorescence and western blot. Cellular functions were determined using CCK-8, colony formation and propidium iodide (PI) staining. Xenograft assay was performed to further verify the effects of formononetin (FM) on TNBC. We found that FM combined therapy suppressed the metastasis of TNBC and increased the overall survival rates of TNBC patients. Moreover, FM suppressed the proliferation and induced mitochondrial damage and apoptosis of TNBC cells. FM increased the expression of the BTB domain and CNC homolog 1 (BACH1) in TNBC tissues as well as cells. However, BACH1 knockdown antagonized the effects of FM and promoted the survival of TNBC cells. FM suppressed the tumor growth of TNBC. Taken together, FM suppressed the aggressiveness of TNBC via BACH1/p53 signaling. Therefore, FM may be an alternative strategy for TNBC.

Study of the in Vivo Functional Role of Mutations in the BTB Domain of the CP190 Protein of Drosophila melanogaster.

The Drosophila transcription factor СР190 is one of the key proteins that determine the activity of housekeeping gene promoters and insulators. CP190 has an N-terminal BTB domain that allows for dimerization. Many of known Drosophila architectural proteins interact with the hydrophobic peptide-binding groove in the BTB domain, which is presumably a mechanisms for recruiting CP190 to regulatory elements. To study the role of the BTB domain in the interaction with architectural proteins, we obtained transgenic flies expressing CP190 variants with mutations in the peptide-binding groove, which disrupts their interaction with architectural proteins. As a result of the studies, it was found that mutations in the BTB domain do not affect binding of the CP190 protein to polytene chromosomes. Thus, our studies confirm the previously obtained data that CP190 is recruited to regulatory elements by several transcription factors interacting, in addition to BTB, with other CP190 domains.

BACH1 encourages ferroptosis by activating KDM4C-mediated COX2 demethylation after cerebral ischemia-reperfusion injury.

It has been confirmed that BTB domain and CNC homologue 1 (BACH1) are involved in ferroptosis-related diseases. However, the function of BACH1 in cerebral ischemia-reperfusion injury (CIRI)-induced ferroptosis remains to be largely unrevealed. First, analysis of differentially expressed genes in CIRI based on the GEO dataset GSE119121 revealed that BACH1 was upregulated in CIRI. BACH1 level was prominently increased in middle cerebral artery occlusion (MCAO)/reperfusion model and oxygen-glucose deprivation/reoxygenation cell model. Further, knock-down of BACH1 markedly reduced iron ion concentration, ROS production, 4-HNE and lipid peroxidation levels and facilitated GSH content, cell viability and protein levels of GPX4 and SLC7A11, while an pcDNA-KDM4C or pcDNA-COX2 combined with BACH1 siRNA could not enhance this effect. Mechanistically, BACH1 bound on the KDM4C promoter to transcriptionally activate its expression. Besides, KDM4C could occupy the promoter locus of the COX2 gene, promoting the COX2 expression by eliminating H3K9me3. Overexpression of KDM4C or COX2 overturned the effects of BACH1 inhibition. In vivo findings displayed that brain infraction, pathological damage and neuronal loss rate in MCAO mice were conspicuously decreased after BACH1 knock-down. This study reveals that BACH1 encourages ferroptosis in neuroblastoma cells and CIRI mouse brain tissues by activating KDM4C-mediated COX2 demethylation.

OsMBTB32, a MATH-BTB domain-containing protein that interacts with OsCUL1s to regulate salt tolerance in rice.

MATH-BTB proteins are involved in a variety of cellular processes that regulate cell homeostasis and developmental processes. Previous studies reported the involvement of BTB proteins in the development of various organs in plants; however, the function of BTB proteins in salt stress is less studied. Here, we found a novel MATH-BTB domain-containing OsMBTB32 protein that was highly expressed in leaf, root, and shoot. The up-regulation of the OsMBTB32 transcript in 2-week-old seedlings under salt stress suggests the significant role of the OsMBTB32 gene in salinity. The OsMBTB32 transgenic seedlings (OE and RNAi) exhibited significant differences in various phenotypes, including plumule, radical, primary root, and shoot length, compared to WT seedlings. We further found that OsCUL1 proteins, particularly OsCUL1-1 and OsCUL1-3, interact with OsMBTB32 and may suppress the function of OsMBTB32 during salt stress. Moreover, OsWRKY42, a homolog of ZmWRKY114 which negatively regulates salt stress in rice, directly binds to the W-box of OsCUL1-1 and OsCUL1-3 promoters to promote the interaction of OsCUL1-1 and OsCUL1-3 with OsMBTB32 protein in rice. The overexpression of OsMBTB32 and OsCUL1-3 further confirmed the function of OsMBTB32 and OsCUL1s in salt tolerance in Arabidopsis. Overall, the findings of the present study provide promising knowledge regarding the MATH-BTB domain-containing proteins and their role in enhancing the growth and development of rice under salt stress.MATH-BTB proteins are involved in a variety of cellular processes that regulate cell homeostasis and developmental processes. Previous studies reported the involvement of BTB proteins in the development of various organs in plants; however, the function of BTB proteins in salt stress is less studied. Here, we found a novel MATH-BTB domain-containing OsMBTB32 protein that was highly expressed in leaf, root, and shoot. The up-regulation of the OsMBTB32 transcript in 2-week-old seedlings under salt stress suggests the significant role of the OsMBTB32 gene in salinity. The OsMBTB32 transgenic seedlings (OE and RNAi) exhibited significant differences in various phenotypes, including plumule, radical, primary root, and shoot length, compared to WT seedlings. We further found that OsCUL1 proteins, particularly OsCUL1-1 and OsCUL1-3, interact with OsMBTB32 and may suppress the function of OsMBTB32 during salt stress. Moreover, OsWRKY42, a homolog of ZmWRKY114 which negatively regulates salt stress in rice, directly binds to the W-box of OsCUL1-1 and OsCUL1-3 promoters to promote the interaction of OsCUL1-1 and OsCUL1-3 with OsMBTB32 protein in rice. The overexpression of OsMBTB32 and OsCUL1-3 further confirmed the function of OsMBTB32 and OsCUL1s in salt tolerance in Arabidopsis. Overall, the findings of the present study provide promising knowledge regarding the MATH-BTB domain-containing proteins and their role in enhancing the growth and development of rice under salt stress.

BTB domains: A structural view of evolution, multimerization, and protein-protein interactions.

Broad-complex, Tramtrack, and Bric-à-brac/poxvirus and zinc finger (BTB/POZ) is a conserved domain found in many eukaryotic proteins with diverse cellular functions. Recent studies revealed its importance in multiple developmental processes as well as in the onset and progression of oncological diseases. Most BTB domains can form multimers and selectively interact with non-BTB proteins. Structural studies of BTB domains delineated the presence of different interfaces involved in various interactions mediated by BTBs and provided a basis for the specific inhibition of distinct protein-interaction interfaces. BTB domains originated early in eukaryotic evolution and progressively adapted their structural elements to perform distinct functions. In this review, we summarize and discuss the structural principles of protein-protein interactions mediated by BTB domains based on the recently published structural data and advances in protein modeling. We propose an update to the structure-based classification of BTB domain families and discuss their evolutionary interconnections.

Ryanodine receptor 2 promotes colorectal cancer metastasis by the ROS/BACH1 axis.

There is no targeted therapy for KRAS proto-oncogene, GTPase (KRAS)-mutant metastatic colorectal cancer (mCRC) because the underlying mechanism remains obscure. Based on bioinformatic analysis, this study aims to elucidate a potential gene target for which an approved drug is available, and to reveal the function as well as the underlying mechanism of the candidate gene. Here, we identified that ryanodine receptor 2 (RyR2) expression was upregulated in KRAS-mutant mCRC, and that this promoted cancer cell metastasis. S107, an approved drug to inhibit calcium release from RyR2 in the clinic, inhibited cancer cell metastasis both in vitro and in vivo. High expression of RyR2 predicts poor survival in our patient cohort. CRC patients with serosa invasion and vascular tumor thrombus are characterized by high RyR2 expression. Analysis of expression profiles upon RyR2 knockdown and inhibition, revealed a set of metastasis-related molecules, and identified BTB domain and CNC homolog 1 (BACH1) as the main transcription factor regulated by RyR2. RyR2 regulates cellular reactive oxygen species (ROS) levels, which activates nuclear factor erythroid 2-related factor 2 (Nrf2; also known as NFE2L2) and HMOX1 expression, and thus BACH1 accumulation. Collectively, this study provides evidence that the RyR2/ROS/BACH1 axis may be a potential intervention target for CRC metastasis.

Increased slow dynamics defines ligandability of BTB domains.

Efficient determination of protein ligandability, or the propensity to bind small-molecules, would greatly facilitate drug development for novel targets. Ligandability is currently assessed using computational methods that typically consider the static structural properties of putative binding sites or by experimental fragment screening. Here, we evaluate ligandability of conserved BTB domains from the cancer-relevant proteins LRF, KAISO, and MIZ1. Using fragment screening, we discover that MIZ1 binds multiple ligands. However, no ligands are uncovered for the structurally related KAISO or LRF. To understand the principles governing ligand-binding by BTB domains, we perform comprehensive NMR-based dynamics studies and find that only the MIZ1 BTB domain exhibits backbone µs-ms time scale motions. Interestingly, residues with elevated dynamics correspond to the binding site of fragment hits and recently defined HUWE1 interaction site. Our data argue that examining protein dynamics using NMR can contribute to identification of cryptic binding sites, and may support prediction of the ligandability of novel challenging targets.

BTB domain and CNC homolog 1 promotes glioma invasion mainly through regulating extracellular matrix and increases ferroptosis sensitivity.

BTB Domain and CNC Homolog 1 (Bach1) has been implicated in cancer progression, particularly in invasion, but little is unknown about its effect on glioma. Here, we confirmed that highly expressed Bach1 prominently promoted glioma invasion. Similar to the reported mechanisms in other tumors, Bach1 upregulation was also correlated with epithelial mesenchymal transition (EMT) in glioma cells. More importantly, proteomic analysis indicated that the main mechanism of Bach1 promoting invasion in glioma involved extracellular matrix (ECM). We further found thatBach1 upregulation was associated with the multiple mechanisms of ECM remodeling in glioma, including increasing the expression and deposition of ECM components, activating TGFBR2-smad2/3 signaling, promoting invadopodia formation and inducing the expression and secretion of MMP2. Meanwhile, Bach1 overexpression increased ferroptosis sensitivity in glioma cells. The ferroptosis inducer (sulfasalazine) obviously suppressed the gliomas with Bach1 upregulation in vitro and in vivo. Overall, Bach1 has a two-faced role in glioma. Highly expressed Bach1 promotes glioma invasion. Conversely, Bach1 upregulation is also a potential indicator of the sensitivity of ferroptosis inducers.

The transcription factor Bach2 negatively regulates murine natural killer cell maturation and function.

BTB domain And CNC Homolog 2 (Bach2) is a transcription repressor that actively participates in T and B lymphocyte development, but it is unknown if Bach2 is also involved in the development of innate immune cells, such as natural killer (NK) cells. Here, we followed the expression of Bach2 during murine NK cell development, finding that it peaked in immature CD27+CD11b+ cells and decreased upon further maturation. Bach2 showed an organ and tissue-specific expression pattern in NK cells. Bach2 expression positively correlated with the expression of transcription factor TCF1 and negatively correlated with genes encoding NK effector molecules and those involved in the cell cycle. Lack of Bach2 expression caused changes in chromatin accessibility of corresponding genes. In the end, Bach2 deficiency resulted in increased proportions of terminally differentiated NK cells with increased production of granzymes and cytokines. NK cell-mediated control of tumor metastasis was also augmented in the absence of Bach2. Therefore, Bach2 is a key checkpoint protein regulating NK terminal maturation.

Remifentanil alleviates hypoxic-ischemic brain damage-induced cognitive impairment via BACH1.

Hypoxia-ischemia (HI) is among the most frequent causes of death and disability in neonates. We aimed here to examine the neuroprotective effects of Remifentanil (RE) and the underlying mechanisms in a rat model of hypoxic-ischemic brain damage (HIBD). We found that RE improved the learning memory ability, reduced neuronal cell damage and apoptosis, reduced inflammation induced by suppressing the expression of BTB domain and CNC homolog 1 (BACH1) in rats with HIBD. BACH1 attenuated the alleviating effect of RE on cognitive impairment in HIBD rats. Moreover, RE inhibited TRAF3 expression by downregulating BACH1, and TRAF3 attenuated the therapeutic effect of RE on cognitive impairment by activating the NF-κB signaling. In conclusion, our findings demonstrated that RE inhibits the expression of BACH1, which in turn inhibits the NF-κB signaling pathway by suppressing TRAF3. RE may be a promising therapeutic agent to attenuate HIBD-induced cognitive impairment.

Three residues in the BTB domain promote a good partnership between NuRD and Thpok.

Among the BTB-ZF transcription factor family, three amino acids in the BTB domain make Thpok unique in repressing cytotoxic lineage-related genes via recruitment of the NuRD chormatin-remodeling complex (see the related Research Article by Gao et al.).

Optimizing Shape Complementarity Enables the Discovery of Potent Tricyclic BCL6 Inhibitors.

To identify new chemical series with enhanced binding affinity to the BTB domain of B-cell lymphoma 6 protein, we targeted a subpocket adjacent to Val18. With no opportunities for strong polar interactions, we focused on attaining close shape complementarity by ring fusion onto our quinolinone lead series. Following exploration of different sized rings, we identified a conformationally restricted core which optimally filled the available space, leading to potent BCL6 inhibitors. Through X-ray structure-guided design, combined with efficient synthetic chemistry to make the resulting novel core structures, a >300-fold improvement in activity was obtained by the addition of seven heavy atoms.

Inhibiting BTB domain and CNC homolog 1 (Bach1) as an alternative to increase Nrf2 activation in chronic diseases.

BTB and CNC homology 1 (Bach1) is a protein that forms nuclear heterodimers with the small musculoaponeurotic fibrosarcoma (sMaf). These bind to genomic DNA, promoting the inhibition of the synthesis of a range of antioxidant enzymes. This heterodimer antagonises the actions of nuclear factor erythroid 2-related factor-2 (Nrf2), a master regulator of cytoprotective responses in the cells. Studies have shown that Nrf2 expression is downregulated and Bach1 expression upregulated in many chronic diseases; hence Nrf2 activators and Bach1 inhibitors need to be investigated for their potential to mitigate inflammation and improve antioxidant responses in the chronic burden of lifestyle diseases, including chronic kidney disease. Thus, this review will discuss the status of Bach1 in such diseases and the use of possible inhibitors as a promising therapeutic approach.

A BTB/POZ domain-containing protein negatively regulates plant immunity in Nicotiana benthamiana.

Plants have evolved immune systems to fight against pathogens. However, it is still largely unknown how the plant immunity is finely regulated. Here we identified a BTB/POZ domain-containing protein, namely NbBTB, which is predicted to be a member of the ubiquitin E3 ligase complex. The NbBTB expression is downregulated upon the oomycete pathogen Phytophthora parasitica infection. Overexpression of NbBTB in Nicotiana benthamiana promoted plant susceptibility to P. parasitica infection, and silencing NbBTB increased plant resistance to P. parasitica, indicating that NbBTB negatively modulates plant basal defense. Interestingly, overexpressing or silencing NbBTB did not affect plant resistance to two bacterial pathogens Ralstonia solanacearum and Pseudomonas syringae, suggesting that NbBTB is specifically involved in basal defense against oomycete pathogen. Expression of NbBTB suppressed hypersensitive response (HR) triggered by avirulence proteins from both R. sonanacearum and P. infestans, and silencing NbBTB showed the opposite effect, indicating that NbBTB negatively regulates effector-triggered immunity (ETI). Protein accumulation of avirulence effectors in NbBTB-silenced plants was significantly enhanced, suggesting that NbBTB is likely to negatively modulate ETI by affecting effector protein accumulation. Together, our results demonstrated that NbBTB is a negative regulator in both plant basal defense and ETI.

Structural basis of Apt48 inhibition of the BCL6 BTB domain.

B cell lymphoma 6 (BCL6) is a transcriptional repressor that is deregulated in diffuse large B cell lymphoma, and the peptide aptamer, Apt48, inhibits BCL6 by an unknown mechanism. We report the crystal structure of BCL6 in complex with an Apt48 peptide, and show that Apt48 binds to a therapeutically uncharacterized region at the bottom of the BCL6 BTB domain. We show that the corepressor binding site of the BTB domain may be divided conceptually into two low-affinity, peptide-binding regions. An upper region, the lateral groove, binds peptides in robust three-dimensional conformations, whereas a lower binding site is permissive to less-specific interactions. We show that, even with little sequence specificity, the interactions of the lower region are required for the high-affinity binding of the SMRT corepressor and other peptides to the BTB domain. This has relevance for the design of new BCL6 inhibitors and for understanding the evolution of corepressor interactions with the BTB domain.

Mutation in the Plasmodium falciparum BTB/POZ Domain of K13 Protein Confers Artemisinin Resistance.

Partial artemisinin resistance, defined in patients as a delayed parasite clearance following artemisinin-based treatment, is conferred by non-synonymous mutations in the Kelch beta-propeller domain of the Plasmodium falciparum k13 (pfk13) gene. Here, we carried out in vitro selection over a 1-year period on a West African P. falciparum strain isolated from Kolle (Mali) under a dose-escalating artemisinin regimen. After 18 cycles of sequential drug pressure, the selected parasites exhibited enhanced survival to dihydroartemisinin in the ring-stage survival assay (RSA0-3h = 9.2%). Sanger and whole-genome sequence analyses identified the PfK13 P413A mutation, localized in the BTB/POZ domain, upstream of the propeller domain. This mutation was sufficient to confer in vitro artemisinin resistance when introduced into the PfK13 coding sequence of the parasite strain Dd2 by CRISPR/Cas9 gene editing. These results together with structural studies of the protein demonstrate that the propeller domain is not the sole in vitro mediator of PfK13-mediated artemisinin resistance, and highlight the importance of monitoring for mutations throughout PfK13.