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.

Structural basis for dimerization quality control.

Most quality control pathways target misfolded proteins to prevent toxic aggregation and neurodegeneration1. Dimerization quality control further improves proteostasis by eliminating complexes of aberrant composition2, but how it detects incorrect subunits remains unknown. Here we provide structural insight into target selection by SCF-FBXL17, a dimerization-quality-control E3 ligase that ubiquitylates and helps to degrade inactive heterodimers of BTB proteins while sparing functional homodimers. We find that SCF-FBXL17 disrupts aberrant BTB dimers that fail to stabilize an intermolecular ß-sheet around a highly divergent ß-strand of the BTB domain. Complex dissociation allows SCF-FBXL17 to wrap around a single BTB domain, resulting in robust ubiquitylation. SCF-FBXL17 therefore probes both shape and complementarity of BTB domains, a mechanism that is well suited to establish quality control of complex composition for recurrent interaction modules.

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.

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.

Control of inflorescence architecture in tomato by BTB/POZ transcriptional regulators.

Plant productivity depends on inflorescences, flower-bearing shoots that originate from the stem cell populations of shoot meristems. Inflorescence architecture determines flower production, which can vary dramatically both between and within species. In tomato plants, formation of multiflowered inflorescences depends on a precisely timed process of meristem maturation mediated by the transcription factor gene TERMINATING FLOWER (TMF), but the underlying mechanism is unknown. We show that TMF protein acts together with homologs of the Arabidopsis BLADE-ON-PETIOLE (BOP) transcriptional cofactors, defined by the conserved BTB (Broad complex, Tramtrack, and Bric-a-brac)/POZ (POX virus and zinc finger) domain. TMF and three tomato BOPs (SlBOPs) interact with themselves and each other, and TMF recruits SlBOPs to the nucleus, suggesting formation of a transcriptional complex. Like TMF, SlBOP gene expression is highest during vegetative and transitional stages of meristem maturation, and CRISPR/Cas9 elimination of SlBOP function causes pleiotropic defects, most notably simplification of inflorescences into single flowers, resembling tmf mutants. Flowering defects are enhanced in higher-order slbop tmf mutants, suggesting that SlBOPs function with additional factors. In support of this, SlBOPs interact with TMF homologs, mutations in which cause phenotypes like slbop mutants. Our findings reveal a new flowering module defined by SlBOP-TMF family interactions that ensures a progressive meristem maturation to promote inflorescence complexity.

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.

The zinc finger and BTB domain containing protein ZBTB20 regulates plasma triglyceride metabolism by repressing lipoprotein lipase gene transcription in hepatocytes.

BACKGROUND AND AIMS: Lipoprotein lipase (LPL) is responsible for the lipolytic processing of triglyceride-rich lipoproteins, the deficiency of which causes severe hypertriglyceridemia. Liver LPL expression is high in suckling rodents but relatively low at adulthood. However, the regulatory mechanism and functional significance of liver LPL expression are incompletely understood. We have established the zinc finger protein ZBTB20 as a critical factor for hepatic lipogenesis. Here, we evaluated the role of ZBTB20 in regulating liver Lpl gene transcription and plasma triglyceride metabolism. APPROACH AND RESULTS: Hepatocyte-specific inactivation of ZBTB20 in mice led to a remarkable increase in LPL expression at the mRNA and protein levels in adult liver, in which LPL protein was mainly localized onto sinusoidal epithelial cells and Kupffer cells. As a result, the LPL activity in postheparin plasma was substantially increased, and postprandial plasma triglyceride clearance was significantly enhanced, whereas plasma triglyceride levels were decreased. The dysregulated liver LPL expression and low plasma triglyceride levels in ZBTB20-deficient mice were normalized by inactivating hepatic LPL expression. ZBTB20 deficiency protected the mice against high-fat diet-induced hyperlipidemia without causing excessive triglyceride accumulation in the liver. Chromatin immunoprecipitation and gel-shift assay studies revealed that ZBTB20 binds to the LPL promoter in the liver. A luciferase reporter assay revealed that ZBTB20 inhibits the transcriptional activity of LPL promoter. The regulation of LPL expression by ZBTB20 is liver-specific under physiological conditions. CONCLUSIONS: Liver ZBTB20 serves as a key regulator of LPL expression and plasma triglyceride metabolism and could be a therapeutic target for hypertriglyceridemia.

Identification of the transcription factor Miz1 as an essential regulator of diphthamide biosynthesis using a CRISPR-mediated genome-wide screen.

Diphthamide is a unique post-translationally modified histidine residue (His715 in all mammals) found only in eukaryotic elongation factor-2 (eEF-2). The biosynthesis of diphthamide represents one of the most complex modifications, executed by protein factors conserved from yeast to humans. Diphthamide is not only essential for normal physiology (such as ensuring fidelity of mRNA translation), but is also exploited by bacterial ADP-ribosylating toxins (e.g., diphtheria toxin) as their molecular target in pathogenesis. Taking advantage of the observation that cells defective in diphthamide biosynthesis are resistant to ADP-ribosylating toxins, in the past four decades, seven essential genes (Dph1 to Dph7) have been identified for diphthamide biosynthesis. These technically unsaturated screens raise the question as to whether additional genes are required for diphthamide biosynthesis. In this study, we performed two independent, saturating, genome-wide CRISPR knockout screens in human cells. These screens identified all previously known Dph genes, as well as further identifying the BTB/POZ domain-containing transcription factor Miz1. We found that Miz1 is absolutely required for diphthamide biosynthesis via its role in the transcriptional regulation of Dph1 expression. Mechanistically, Miz1 binds to the Dph1 proximal promoter via an evolutionarily conserved consensus binding site to activate Dph1 transcription. Therefore, this work demonstrates that Dph1-7, along with the newly identified Miz1 transcription factor, are likely to represent the essential protein factors required for diphthamide modification on eEF2.

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.

The BTB-domain transcription factor ZBTB2 recruits chromatin remodelers and a histone chaperone during the exit from pluripotency.

Transcription factors (TFs) harboring broad-complex, tramtrack, and bric-a-brac (BTB) domains play important roles in development and disease. These BTB domains are thought to recruit transcriptional modulators to target DNA regions. However, a systematic molecular understanding of the mechanism of action of this TF family is lacking. Here, we identify the zinc finger BTB-TF Zbtb2 from a genetic screen for regulators of exit from pluripotency and demonstrate that its absence perturbs embryonic stem cell differentiation and the gene expression dynamics underlying peri-implantation development. We show that ZBTB2 binds the chromatin remodeler Ep400 to mediate downstream transcription. Independently, the BTB domain directly interacts with nucleosome remodeling and deacetylase and histone chaperone histone regulator A. Nucleosome remodeling and deacetylase recruitment is a common feature of BTB TFs, and based on phylogenetic analysis, we propose that this is a conserved evolutionary property. Binding to UBN2, in contrast, is specific to ZBTB2 and requires a C-terminal extension of the BTB domain. Taken together, this study identifies a BTB-domain TF that recruits chromatin modifiers and a histone chaperone during a developmental cell state transition and defines unique and shared molecular functions of the BTB-domain TF family.

The uncharacterized SANT and BTB domain-containing protein SANBR inhibits class switch recombination.

Class switch recombination (CSR) is the process by which B cells switch production from IgM/IgD to other immunoglobulin isotypes, enabling them to mount an effective immune response against pathogens. Timely resolution of CSR prevents damage due to an uncontrolled and prolonged immune response. While many positive regulators of CSR have been described, negative regulators of CSR are relatively unknown. Using an shRNA library screen targeting more than 28,000 genes in a mouse B cell line, we have identified a novel, uncharacterized protein of 82kD (KIAA1841, NM_027860), which we have named SANBR (SANT and BTB domain regulator of CSR), as a negative regulator of CSR. The purified, recombinant BTB domain of SANBR exhibited characteristic properties such as homodimerization and interaction with corepressor proteins, including HDAC and SMRT. Overexpression of SANBR inhibited CSR in primary mouse splenic B cells, and inhibition of CSR is dependent on the BTB domain while the SANT domain is largely dispensable. Thus, we have identified a new member of the BTB family that serves as a negative regulator of CSR. Future investigations to identify transcriptional targets of SANBR in B cells will reveal further insights into the specific mechanisms by which SANBR regulates CSR as well as fundamental gene regulatory activities of this protein.

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.

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.

DUBR suppresses migration and invasion of human lung adenocarcinoma cells via ZBTB11-mediated inhibition of oxidative phosphorylation.

Long noncoding RNAs (lncRNAs) are involved in a variety of cancers, but the role of LncRNA DUBR in lung adenocarcinoma (LUAD), the most prevalent form of lung cancer, remains unclear. In this study we investigated the expression of DUBR in LUAD to ascertain its association with the clinical pathology and prognosis of LUAD. Analysis of mRNA expression in The Cancer Genome Atlas (TCGA) LUAD database and in-house LUAD cohort (n = 94) showed that DUBR was significantly downregulated in LUAD, and was associated with poor prognosis. In LUAD cell lines (H1975, A549), overexpression of DUBR significantly suppressed the migration and invasion of the LUAD cells. We demonstrated that c-Myc could bind to the promoter of DUBR, and transcriptionally suppressed its expression. Knockdown of c-Myc almost completely blocked the invasion and migration of LUAD cells, whereas knockdown of DUBR partially rescued c-Myc-knockdown suppressed cell migration and invasion. Furthermore, DUBR overexpression significantly increased the expression of a downstream protein of DUBR, zinc finger, and BTB domain containing 11 (ZBTB11), in H1975 and A549 cells; knockdown of ZBTB11 partially rescued the DUBR-overexpression suppressed cell migration and invasion; knockdown of c-Myc significantly upregulated the expression of ZBTB11 in LUAD cells. Finally, we revealed that DUBR/ZBTB11 axis suppressed oxidative phosphorylation in LUAD cells. In short, we demonstrate that c-Myc/DUBR/ZBTB11 axis suppresses migration and invasion of LUAD by attenuating cell oxidative phosphorylation, which provides new insights into the regulatory mechanism of DUBR.

A novel missense variant in CUL3 shows altered binding ability to BTB-adaptor proteins leading to diverse phenotypes of CUL3-related disorders.

CUL3 forms Cullin-Ring ubiquitin ligases (CRL) with Ring-box protein and BTB-adaptor proteins. A variety of BTB-adaptor proteins have been reported to interact with the N-terminus of CUL3, which makes it possible to recognize various substrates for degradation. Regarding the association of CUL3 with neurodevelopmental disorders, a recent study reported three patients with global developmental delay, who carried de novo variants in CUL3. Here, we describe a novel de novo CUL3 variant (c.158G > A, p.Ser53Asn) identified in a patient with global developmental delay, who presented some novel dysmorphic features, including macrocephaly, characteristic facial features, and cutis marmorata. Immunoprecipitation and immunoblot analyses identified significantly weaker binding ability to some BTB proteins in CUL3-S53N compared to wild-type. Interestingly, label-free quantification proteomics analysis of samples immunoprecipitated by CUL3-S53N showed a significantly decreased interaction with some BTB proteins, while almost equal interaction or significantly increased interaction was observed with other BTB proteins. The binding between CUL3 and BTB proteins is essential for CRL substrate recognition, and alteration of their interaction is thought to result in the quantitative alteration in substrate proteins. It is possible that the difference of dysmorphic features between the present case and previously reported cases is caused by the distinctive effect of each CUL3 variant on substrate proteins. The clinical information of the present case will expand the picture of CUL3-related global developmental disorders, and subsequent cell biological analysis of the novel mutation will provide insight into the underlying molecular mechanism of how CUL3 pathogenic variants cause neurological disorders.

The BTB/POZ domain protein GmBTB/POZ promotes the ubiquitination and degradation of the soybean AP2/ERF-like transcription factor GmAP2 to regulate the defense response to Phytophthora sojae.

Phytophthora root and stem rot in soybean (Glycine max) is a destructive disease worldwide, and hence improving crop resistance to the causal pathogen, P. sojae, is a major target for breeders. However, it remains largely unclear how the pathogen regulates the various affected signaling pathways in the host, which consist of complex networks including key transcription factors and their targets. We have previously demonstrated that GmBTB/POZ enhances soybean resistance to P. sojae and the associated defense response. Here, we demonstrate that GmBTB/POZ interacts with the transcription factor GmAP2 and promotes its ubiquitination. GmAP2-RNAi transgenic soybean hairy roots exhibited enhanced resistance to P. sojae, whereas roots overexpressing GmAP2 showed hypersensitivity. GmWRKY33 was identified as a target of GmAP2, which represses its expression by directly binding to the promoter. GmWRKY33 acts as a positive regulator in the response of soybean to P. sojae. Overexpression of GmBTB/POZ released the GmAP2-regulated suppression of GmWRKY33 in hairy roots overexpressing GmAP2 and increased their resistance to P. sojae. Taken together, our results indicate that GmBTB/POZ-GmAP2 modulation of the P. sojae resistance response forms a novel regulatory mechanism, which putatively regulates the downstream target gene GmWRKY33 in soybean.

BTB domain-containing 7 predicts low recurrence and suppresses tumor progression by deactivating Notch1 signaling in breast cancer.

PURPOSE: BTB domain-containing 7 (BTBD7) has been found to regulate epithelial tissue remodeling and branched organ formation and has been reported to modulate the biological behavior of several cancers. However, its role in breast cancer has not been identified. This study investigated the biological role and prognostic value of BTBD7 in breast cancer. METHODS: We identified the BTBD7 expression pattern using the GENT2 database and assessed its expression in breast cancer tissue and cell lines using quantitative reverse transcription polymerase chain reaction, western blot, and immunohistochemistry. We conducted a clinical relevance and survival analysis on a cohort of 121 breast cancer cases from our follow-up and validated it in a Kaplan-Meier plotter. The gain-loss effect of BTBD7 on cell proliferation, invasion, and migration was detected in vitro. We employed a xenograft mouse metastatic model for in vivo validation and performed a Cignal Finder Cancer 10-Pathway Reporter Array, western blot, immunofluorescence, Cell Counting Kit-8, and transwell invasion/migration assays to analyze the potential mechanism. RESULTS: BTBD7 was downregulated in human breast cancer cell lines and tissues. Decreased BTBD7 expression correlated with a positive lymph node status, lymphovascular invasion, and TNM stage, while high BTBD7 expression correlated with low breast cancer recurrence. BTBD7 suppressed cell proliferation, invasion/migration, and tumor metastasis in breast cancer. The mechanism studied suggested that the inhibitory role of BTBD7 was through the deactivation of Notch1 signaling in breast cancer. CONCLUSION: BTBD7 suppresses tumor progression, and its high expression correlates with low recurrence in breast cancer.

Vaccinia Virus BBK E3 Ligase Adaptor A55 Targets Importin-Dependent NF-κB Activation and Inhibits CD8+ T-Cell Memory.

Viral infection of cells is sensed by pathogen recognition receptors that trigger an antiviral innate immune response, and consequently viruses have evolved countermeasures. Vaccinia virus (VACV) evades the host immune response by expressing scores of immunomodulatory proteins. One family of VACV proteins are the BTB-BACK (broad-complex, tram-trac, and bric-a-brac [BTB] and C-terminal Kelch [BACK]) domain-containing, Kelch-like (BBK) family of predicted cullin-3 E3 ligase adaptors: A55, C2, and F3. Previous studies demonstrated that gene A55R encodes a protein that is nonessential for VACV replication yet affects viral virulence in vivo Here, we report that A55 is an NF-κB inhibitor acting downstream of IκBα degradation, preventing gene transcription and cytokine secretion in response to cytokine stimulation. A55 targets the host importin α1 (KPNA2), acting to reduce p65 binding and its nuclear translocation. Interestingly, while A55 was confirmed to coprecipitate with cullin-3 in a BTB-dependent manner, its NF-κB inhibitory activity mapped to the Kelch domain, which alone is sufficient to coprecipitate with KPNA2 and inhibit NF-κB signaling. Intradermal infection of mice with a virus lacking A55R (vΔA55) increased VACV-specific CD8+ T-cell proliferation, activation, and cytotoxicity in comparison to levels of the wild-type (WT) virus. Furthermore, immunization with vΔA55 induced increased protection to intranasal VACV challenge compared to the level with control viruses. In summary, this report describes the first target of a poxvirus-encoded BBK protein and a novel mechanism for DNA virus immune evasion, resulting in increased CD8+ T-cell memory and a more immunogenic vaccine.IMPORTANCE NF-κB is a critical transcription factor in the innate immune response to infection and in shaping adaptive immunity. The identification of host and virus proteins that modulate the induction of immunological memory is important for improving virus-based vaccine design and efficacy. In viruses, the expression of BTB-BACK Kelch-like (BBK) proteins is restricted to poxviruses and conserved within them, indicating the importance of these proteins for these medically important viruses. Using vaccinia virus (VACV), the smallpox vaccine, we report that the VACV BBK protein A55 dysregulates NF-κB signaling by disrupting the p65-importin interaction, thus preventing NF-κB translocation and blocking NF-κB-dependent gene transcription. Infection with VACV lacking A55 induces increased VACV-specific CD8+ T-cell memory and better protection against VACV challenge. Studying viral immunomodulators therefore expands not only our understanding of viral pathogenesis and immune evasion strategies but also of the immune signaling cascades controlling antiviral immunity and the development of immune memory.

Generation and analysis of a mouse model of pseudohypoaldosteronism type II caused by KLHL3 mutation in BTB domain.

The Kelch-like 3 ( KLHL3) mutations contributed to the most common causative genes in patients with pseudohypoaldosteronism type II (PHAII); however, the molecular mechanisms of PHAII-causing mutations in BTB domain of KLHL3 in vivo have not been investigated. We generated and analyzed Klhl3 knock-in (KI) mice carrying a missense M131V mutation in the BTB domain (corresponding to human KLHL3 M78V mutation). Klhl3M131V/+ KI mice exhibited typical PHAII phenotype with an exaggerated diuretic response to hydrochlorothiazide. Their kidney tissues showed an unchanged KLHL3, decreased cullin 3 (Cul3), and increased with-no-lysine kinases (WNKs) WNK1 and WNK4 along with an enhanced downstream ste20-related proline/alanine-rich kinase/oxidative stress response kinase 1-N(K)CC phosphorylation. Their Cul3 protein in the cytosol of distal convoluted tubule cells was also significantly attenuated on immunogold-labeling electron microscopy. In microdissected renal tubules, Klhl3M131V/+ KI mice expressed high levels of Wnk4 mRNA in the distal nephron. In vitro coimmunoprecipitation showed the KLHL3 BTB domain mutation retained intact interaction with WNKs but reduced binding to Cul3, thus leading to the increased abundance of total WNKs. In summary, Klhl3M131V/+ KI mice feature typical PHAII with a simultaneous increase of WNK1 and WNK4 through the impaired KLHL3 BTB domain binding to Cul3.-Lin, C.-M., Cheng, C.-J., Yang, S.-S., Tseng, M.-H., Yen, M.-T., Sung, C.-C., Lin, S.-H. Generation and analysis of a mouse model of pseudohypoaldosteronism type II caused by KLHL3 mutation in BTB domain.