BTB Proteins Research Articles

Polymerization of ZBTB transcription factors regulates chromatin occupancy

BCL6, an oncogenic transcription factor (TF), forms polymers in the presence of a small-molecule molecular glue that stabilizes a complementary interface between homodimers of BCL6's broad-complex, tramtrack, and bric-à-brac (BTB) domain. The BTB domains of other proteins, including a large class of TFs, have similar architectures and symmetries, raising the possibility that additional BTB proteins self-assemble into higher-order structures. Here, we surveyed 189 human BTB proteins with a cellular fluorescent reporter assay and identified 18 ZBTB TFs that show evidence of polymerization. Through biochemical and cryoelectron microscopy (cryo-EM) studies, we demonstrate that these ZBTB TFs polymerize into filaments. We found that BTB-domain-mediated polymerization of ZBTB TFs enhances chromatin occupancy within regions containing homotypic clusters of TF binding sites, leading to repression of target genes. Our results reveal a role of higher-order structures in regulating ZBTB TFs and suggest an underappreciated role for TF polymerization in modulating gene expression.

Calmodulin-like protein MdCML15 interacts with MdBT2 to modulate iron homeostasis in apple.

BTB and TAZ domain proteins (BTs) function as specialized adaptors facilitating substrate recognition of the CUL3-RING ubiquitin ligase (CRL3) complex that targets proteins for ubiquitination in reaction to diverse pressures. Nonetheless, knowledge of the molecular mechanisms by which the apple scaffold protein MdBT2 responds to external and internal signals is limited. Here we demonstrate that a putative Ca 2+ sensor, calmodulin-like 15 (MdCML15), acts as an upstream regulator of MdBT2 to negatively modulate its functions in plasma membrane H+-ATPase regulation and iron deficiency tolerance. MdCML15 was identified to be substantially linked to MdBT2, and to result in the ubiquitination and degradation of the MdBT2 target protein MdbHLH104. Consequently, MdCML15 repressed the MdbHLH104 target, MdAHA8's expression, reducing levels of a specific membrane H+-ATPase. Finally, the phenotype of transgenic apple plantlets and calli demonstrated that MdCML15 modulates membrane H+-ATPase-produced rhizosphere pH lowering alongside iron homeostasis through an MdCML15-MdBT2-MdbHLH104-MdAHA8 pathway. Our results provide new insights into the relationship between Ca2+ signaling and iron homeostasis.

BTB protein family and human breast cancer: signaling pathways and clinical progress.

Breast cancer is considered the number one killer of women both in China and abroad, and the leading cause of cancer death. It severely affects female health-related quality of life. Broad-complex, tramtrack, bric à brac (BTB) protein family was first discovered in drosophila as early as in 1993 by Godt D and peers, since then, more family members and their critical biological functions were uncovered. Moreover, researchers around the world have recently demonstrated that numerous signaling pathways connect BTB family members and human breast cancer. In this review, we critically discuss these findings regarding the essential mechanisms and functions of the BTB protein family in mediating the organic processes of human breast cancer. Meanwhile, we summarize the signaling pathways the BTB protein family participates in. And we address that BTB proteins regulate the growth, apoptosis, and other behaviors of breast cancer cells. We also point out the future directions for further studies in this field. The relevant onlineliteratures have been reviewed for this article. This review could offer an update on novel molecular targets for treating human breast cancer and new insights into BTB protein family research.

OsBBX19-OsBTB97/OsBBX11 module regulates spikelet development and yield production in rice

Spikelet and floral-related organs are important agronomic traits for rice grain yield. BTB (broad-complex, tram track, and bric-abrac) proteins control various developmental functions in plants; however, the molecular mechanism of BTB proteins underlying grain development and yield production is still unknown. Here, we evaluated the molecular mechanism of a previously unrecognized functional gene, namely OsBTB97 that regulates the floral and spikelet-related organs which greatly affect the final grain yield. We found that the knockdown of the OsBTB97 gene had significant impacts on the development of spikelet-related organs and grain size, resulting in a decrease in yield, by altering the transcript levels of various spikelet- and grain-related genes. Furthermore, we found that the knockout mutants of two BBX genes, OsBBX11 and OsBBX19, which interact with the OsBTB97 protein at translation and transcriptional level, respectively, displayed lower OsBTB97 expression, suggesting the genetic relationship between the BTB protein and the BBX transcription factors in rice. Taken together, our study dissects the function of the novel OsBTB97 by interacting with two BBX proteins and an OsBBX19-OsBTB97/OsBBX11 module might function in the spikelet development and seed production in rice. The outcome of the present study provides promising knowledge about BTB proteins in the improvement of crop production in plants.

Pathophysiological role of BACH transcription factors in digestive system diseases.

BTB and CNC homologous (BACH) proteins, including BACH1 and BACH2, are transcription factors that are widely expressed in human tissues. BACH proteins form heterodimers with small musculoaponeurotic fibrosarcoma (MAF) proteins to suppress the transcription of target genes. Furthermore, BACH1 promotes the transcription of target genes. BACH proteins regulate physiological processes, such as the differentiation of B cells and T cells, mitochondrial function, and heme homeostasis as well as pathogenesis related to inflammation, oxidative-stress damage caused by drugs, toxicants, or infections; autoimmunity disorders; and cancer angiogenesis, epithelial-mesenchymal transition, chemotherapy resistance, progression, and metabolism. In this review, we discuss the function of BACH proteins in the digestive system, including the liver, gallbladder, esophagus, stomach, small and large intestines, and pancreas. BACH proteins directly target genes or indirectly regulate downstream molecules to promote or inhibit biological phenomena such as inflammation, tumor angiogenesis, and epithelial-mesenchymal transition. BACH proteins are also regulated by proteins, miRNAs, LncRNAs, labile iron, and positive and negative feedback. Additionally, we summarize a list of regulators targeting these proteins. Our review provides a reference for future studies on targeted drugs in digestive diseases.

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 highly interactive BTB domain targeting other functional domains to diversify the function of BTB proteins in rice growth and development

The BTB (broad-complex, tram track, and bric-abrac) proteins are involved in developmental processes, biotic, and abiotic stress responses in various plants, but the molecular basis of protein interactions is yet to be investiagted in rice. In this study, the identified BTB proteins were divided into BTB-TAZ, MATH-BTB, BTB-NPH, BTB-ANK, BTB-Skp, BTB-DUF, and BTB-TPR subfamilies based on the additional functional domains found together with the BTB domain at N- and C-terminal as well. This suggesting that the extension region at both terminal sites could play a vital role in the BTB gene family expansion in plants. The yeast two-hybrid system, firefly luciferase complementation imaging (LCI) assay and bimolecular fluorescence complementation (BiFC) assay further confirmed that BTB proteins interact with several other proteins to perform a certain developmental process in plants. The overexpression of BTB genes of each subfamily in Arabidopsis revealed that BTB genes including OsBTB4, OsBTB8, OsBTB64, OsBTB62, OsBTB138, and OsBTB147, containing certain additional functional domains, could play a potential role in the early flowering, branching, leaf, and silique development. Thus we concluded that the presence of other functional domains such as TAZ, SKP, DUF, ANK, NPH, BACK, PQQ, and MATH could be the factor driving the diverse functions of BTB proteins in plant biology.

Regulation of Rho GTPases in the Vasculature by Cullin3-Based E3 Ligase Complexes.

Cullin3-based ubiquitin E3 ligases induce ubiquitination of substrates leading to their proteasomal or lysosomal degradation. BTB proteins serve as adaptors by binding to Cullin3 and recruiting substrate proteins, which enables specific recognition of a broad spectrum of targets. Hence, Cullin3 and its adaptors are involved in myriad cellular processes and organ functions. Cullin3-based ubiquitin E3 ligase complexes target small GTPases of the Rho subfamily, which are key regulators of cytoskeletal dynamics and cell adhesion. In this mini review, we discuss recent insights in Cullin3-mediated regulation of Rho GTPases and their impact on cellular function and disease. Intriguingly, upstream regulators of Rho GTPases are targeted by Cullin3 complexes as well. Thus, Rho GTPase signaling is regulated by Cullin3 on multiple levels. In addition, we address current knowledge of Cullin3 in regulating vascular function, focusing on its prominent role in endothelial barrier function, angiogenesis and the regulation of blood pressure.

Human CTCF Interacts with Drosophila CP190, but not with Kaiso.

Human CTCF (hCTCF) is a major architectural protein in mammals. In Drosophila, the CTCF homologue (dCTCF) interacts with the BTB domain of the CP190 protein, which is involved in the establishment of open chromatin and activity of insulators. Previously, it was shown that the BTB protein Kaiso interacts with hCTCF and regulates its activity. We have carried out a detailed study of the interaction between these proteins in the yeast two-hybrid assay. Surprisingly, Kaiso did not interact with hCTCF and its Drosophila homologue. On the other hand, CP190 interacted with the C-terminus of hCTCF. The results obtained demonstrate that the interaction between CTCF and CP190 proteins is highly conserved. It is likely that humans have other BTB proteins that perform the functions described for the Drosophila CP190.

Structural basis for dimerization quality control.

Most quality control pathways target misfolded proteins to prevent toxic aggregation and neurodegeneration 1. Dimerization quality control (DQC) further improves proteostasis by eliminating complexes of aberrant composition 2, yet how it detects incorrect subunits is still unknown. Here, we provide structural insight into target selection by SCFFBXL17, a DQC E3 ligase that ubiquitylates and helps degrade inactive heterodimers of BTB proteins, while sparing functional homodimers. We find that SCFFBXL17 disrupts aberrant BTB dimers that fail to stabilize an intermolecular β-sheet around a highly divergent β-strand of the BTB domain. Complex dissociation allows SCFFBXL17 to wrap around a single BTB domain for robust ubiquitylation. SCFFBXL17 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.

Cullin 3 and Its Role in Tumorigenesis.

Cullin 3 (Cul3) family of ubiquitin ligases comprises three components, the RING finger protein RBX1, the Cul3 scaffold, and a Bric-a-brac/Tramtrack/Broad complex (BTB) protein. The BTB protein serves as a bridge to connect Cul3 to substrate and is functionally equivalent to the combination of substrate adaptor and linker in other Cullin complexes. Human genome encodes for ~180 BTB proteins, implying a broad spectrum of ubiquitination signals and substrate repertoire. Accordingly, Cul3 ubiquitin ligases are involved in diverse cellular processes, including cell division, differentiation, cytoskeleton remodeling, stress responses, and nerve cell functions. Emerging evidence has pointed to the prominent role of Cul3 ubiquitin ligases in cancer. This chapter will describe recent advances on the roles of Cul3 E3 ligase complexes in regulating various cancer hallmarks and therapeutic responses and the mutation/dysregulation of Cul3 substrate adaptors in cancer. In particular, we will focus on several extensively studied substrate adaptors, such as Keap1, SPOP, KLHL20, and LZTR1, and will also discuss other recently identified Cul3 adaptors with oncogenic or tumor-suppressive functions. We conclude that Cul3 ubiquitin ligases represent master regulators of human malignancies and highlight the importance of developing modulating agents for oncogenic/tumor-suppressive Cul3 E3 ligase complexes to prevent or intervene tumorigenesis.

Molecular basis of cullin-3 (Cul3) ubiquitin ligase subversion by vaccinia virus protein A55

BTB–Kelch proteins are substrate-specific adaptors for cullin-3 (Cul3) RING-box–based E3 ubiquitin ligases, mediating protein ubiquitylation for subsequent proteasomal degradation. Vaccinia virus encodes three BTB–Kelch proteins: A55, C2, and F3. Viruses lacking A55 or C2 have altered cytopathic effects in cultured cells and altered pathology in vivo. Previous studies have shown that the ectromelia virus orthologue of A55 interacts with Cul3 in cells. We report that the N-terminal BTB-BACK (BB) domain of A55 binds directly to the Cul3 N-terminal domain (Cul3–NTD), forming a 2:2 complex in solution. We solved the structure of an A55BB/Cul3–NTD complex from anisotropic crystals diffracting to 2.3/3.7 Å resolution in the best/worst direction, revealing that the overall interaction and binding interface closely resemble the structures of cellular BTB/Cul3–NTD complexes, despite low sequence identity between A55 and cellular BTB domains. Surprisingly, despite this structural similarity, the affinity of Cul3–NTD for A55BB was stronger than for cellular BTB proteins. Glutamate substitution of the A55 residue Ile-48, adjacent to the canonical φX(D/E) Cul3-binding motif, reduced affinity of A55BB for Cul3–NTD by at least 2 orders of magnitude. Moreover, Ile-48 and the φX(D/E) motif are conserved in A55 orthologues from other poxviruses, but not in the vaccinia virus proteins C2 or F3. The high-affinity interaction between A55BB and Cul3–NTD suggests that, in addition to directing the Cul3–RING E3 ligase complex to degrade cellular/viral target proteins that are normally unaffected, A55 may also sequester Cul3 from cellular adaptor proteins, thereby protecting substrates of these cellular adaptors from ubiquitylation and degradation.

BACH transcription factors in innate and adaptive immunity.

BTB and CNC homology (BACH) proteins are transcriptional repressors of the basic region leucine zipper (bZIP) transcription factor family. Recent studies indicate widespread roles of BACH proteins in controlling the development and function of the innate and adaptive immune systems, including the differentiation of effector and memory cells of the B and T cell lineages, CD4+ regulatory T cells and macrophages. Here, we emphasize similarities at a molecular level in the cell-type-specific activities of BACH factors, proposing that competitive interactions of BACH proteins with transcriptional activators of the bZIP family form a common mechanistic theme underlying their diverse actions. The findings contribute to a general understanding of how transcriptional repressors shape lineage commitment and cell-type-specific functions through repression of alternative lineage programmes.

OsCUL3a Negatively Regulates Cell Death and Immunity by Degrading OsNPR1 in Rice.

Cullin3-based RING E3 ubiquitin ligases (CRL3), composed of Cullin3 (CUL3), RBX1, and BTB proteins, are involved in plant immunity, but the function of CUL3 in the process is largely unknown. Here, we show that rice (Oryza sativa) OsCUL3a is important for the regulation of cell death and immunity. The rice lesion mimic mutant oscul3a displays a significant increase in the accumulation of flg22- and chitin-induced reactive oxygen species, and in pathogenesis-related gene expression as well as resistance to Magnaporthe oryzae and Xanthomonas oryzae pv oryzae. We cloned the OsCUL3a gene via a map-based strategy and found that the lesion mimic phenotype of oscul3a is associated with the early termination of OsCUL3a protein. Interaction assays showed that OsCUL3a interacts with both OsRBX1a and OsRBX1b to form a multisubunit CRL in rice. Strikingly, OsCUL3a interacts with and degrades OsNPR1, which acts as a positive regulator of cell death in rice. Accumulation of OsNPR1 protein is greater in the oscul3a mutant than in the wild type. Furthermore, the oscul3a osnpr1 double mutant does not exhibit the lesion mimic phenotype of the oscul3a mutant. Our data demonstrate that OsCUL3a negatively regulates cell death and immunity by degrading OsNPR1 in rice.

Broad-complex, tramtrack, and bric-à-brac (BTB) proteins: Critical regulators of development.

Broad-complex, Tramtrack, and Bric-à-brac/poxvirus and zinc finger (BTB/POZ) family proteins are a diverse family of proteins that are characterized by the presence of a common protein-protein interaction domain, known as the BTB domain. BTB proteins have been identified in poxviruses and many eukaryotes, and have diverse functions, ranging from transcriptional regulation and chromatin remodeling to protein degradation and cytoskeletal regulation. Specificity of function is determined in part by additional domains present in BTB family proteins, as well as by interaction partners. Studies of BTB proteins in Drosophila and mammalian systems have revealed the importance of these genes in multiple developmental contexts, as well as in cancer and neurological and musculoskeletal diseases. In this review, we discuss the functions of BTB/POZ proteins during development with an emphasis on BTB-zinc finger (BTB-ZF) proteins, which play critical roles in transcriptional regulation and chromatin remodeling.

Sublethal effects of zinc oxide nanoparticles on male reproductive cells

Environmental exposure to nanomaterials is inevitable as nanomaterials become part of our daily life, and as a result, nanotoxicity research is gaining attention. Most investigators focused on the effects of zinc oxide nanoparticles (ZnO NPs) on human health, while limited information was available on the male reproductive system. Herein, mouse Sertoli cell line (TM-4) and spermatocyte cell line (GC2-spd) were used as in vitro models to explore the reproductive effects of ZnO NPs at sublethal dose and its underlying mechanisms. Cells were treated with different concentrations of ZnO NPs. By cell viability assay, a dose of 8μg/mL was found as a sublethal dose and increased the ROS levels in both cells. The decreased glutathione level and increased MDA level were also found in ZnO NPs treated group. In TM4 cells, the expressions of BTB proteins (ZO-1, occludin, claudin-5, and connexin-43) were lower in the ZnO NPs group. The increased cell permeability and increased TNF-α secretion were also observed in ZnO NPs group. In GC2-spd cells, S phase arrest and DNA damage occurred in ZnO NPs group, which could be partially rescued by NAC. Our findings demonstrated that exposure to ZnO NPs induced ROS generation, caused DNA damage of germ cells, and down-regulated the expression of BTB proteins in Sertoli cells which could compromise the integrity of the blood-testis barrier. All these contributed to the male reproductive cytotoxic effects of ZnO NPs that could be partially rescued by anti-oxidants.

Cullin 3 Ubiquitin Ligases in Cancer Biology: Functions and Therapeutic Implications.

Cullin-RING ubiquitin ligases are the largest E3 ligase family in eukaryotes and are multiprotein complexes. In these complexes, the Cullin protein serves as a scaffold to connect two functional modules of the ligases, the catalytic subunit and substrate-binding subunit. To date, eight members of the Cullin family proteins have been identified. In the Cul3 ubiquitin ligases, Bric-a-brac/Tramtrack/Broad complex (BTB) domain-containing proteins function as a bridge to connect Cul3 and substrates. While the BTB domain is responsible for Cul3 binding, these proteins usually contain an additional domain for substrate interaction, such as MATH, kelch, Zn finger, and PAM, Highwire, and RPM-1 (PHR domain). With the existence of a large number of BTB proteins in human, the Cul3 ubiquitin ligases ubiquitinate a wide range of substrates involving in diverse cellular functions. In this review, we will discuss recent advances on the functions of Cul3 ubiquitin ligases in cancer development, progression, and therapeutic response and the dysregulation of Cul3-mediated ubiquitination events in human malignancies. In particular, we will focus on three Cul3 substrate adaptors, kelch-like ECH-associated protein (Keap1), kelch-like family member 20 (KLHL20), and speckle type BTB/POZ protein (SPOP), with the intent to highlight novel targets in cancer therapy.

Expression profile of a Caenorhabditis elegans model of adult neuronal ceroid lipofuscinosis reveals down regulation of ubiquitin E3 ligase components.

Cysteine string protein (CSP) is a chaperone of the Dnaj/Hsp40 family of proteins and is essential for synaptic maintenance. Mutations in the human gene encoding CSP, DNAJC5, cause adult neuronal ceroid lipofucinosis (ANCL) which is characterised by progressive dementia, movement disorders, seizures and premature death. CSP null models in mice, flies and worms have been shown to also exhibit similar neurodegenerative phenotypes. Here we have explored the mechanisms underlying ANCL disease progression using Caenorhaditis elegans mutant strains of dnj-14, the worm orthologue of DNAJC5. Transcriptional profiling of these mutants compared to control strains revealed a broad down-regulation of ubiquitin proteasome system (UPS)-related genes, in particular, components of multimeric RING E3 ubiquitin ligases including F-Box, SKR and BTB proteins. These data were supported by the observation that dnj-14 mutant worm strains expressing a GFP-tagged ubiquitin fusion degradation substrate exhibited decreased ubiquitylated protein degradation. The results indicate that disruption of an essential synaptic chaperone leads to changes in expression levels of UPS-related proteins which has a knock-on effect on overall protein degradation in C. elegans. The specific over-representation of E3 ubiquitin ligase components revealed in our study, suggests that proteins and complexes upstream of the proteasome itself may be beneficial therapeutic targets.

Proteasome-mediated degradation of FRIGIDA modulates flowering time in Arabidopsis during vernalization.

Winter-annual accessions of Arabidopsis thaliana require either exposure to cold stress or vernalization to initiate flowering via FRIGIDA (FRI). FRI acts as a scaffold protein to recruit several chromatin modifiers that epigenetically modify flowering genes. Here, we report that proteasome-mediated FRI degradation regulates flowering during vernalization in Arabidopsis. Our genetic and biochemical experiments demonstrate that FRI directly interacts with the BTB (Bric-a-Brac/Tramtrack/Broad Complex) proteins LIGHT-RESPONSE BTB1 (LRB1) and LRB2 as well as the CULLIN3A (CUL3A) ubiquitin-E3 ligase in vitro and in vivo, leading to proteasomal degradation of FRI during vernalization. The degradation of FRI is accompanied by an increase in the levels of the long noncoding RNA ColdAIR, which reduces the level of histone H3Lys4 trimethylation (H3K4me3) in FLOWERING LOCUS C chromatin to promote flowering. Furthermore, we found that the cold-induced WRKY34 transcription factor binds to the W-box in the promoter region of CUL3A to modulate CUL3A expression. Deficiency of WRKY34 suppressed CUL3A transcription to enhance FRI protein stability and led to late flowering after vernalization. Conversely, overexpression of WRK34 promoted FRI degradation and early flowering through inducing CUL3A accumulation. Together, these data suggest that WRKY34-induced and CUL3A-dependent proteolysis of FRI modulate flowering in response to vernalization.

KCTD proteins as Cullin3 E3 Ligase adapters

Cullin3 (Cul3) is an ubiquitin E3 ligase responsible for catalyzing the transfer of an ubiquitin moiety from an E2 enzyme to a target substrate protein. The C-terminal region of Cul3 binds RBX1/E2-ubiquitin, while, the N-terminal region interacts with various BTB domain proteins which serve as substrate adaptors. Previously, our group determined the crystal structures of the homodimeric BTB proteins SPOP and KLHL3 in complex with the N-terminal domain of Cul3, revealing the determinants responsible for the BTB/Cul3 interaction [1, 2]. A second class of BTB-domain containing proteins, the KCTD proteins, are also Cul3 substrate adaptors but these do not share many of the previously determined features for Cul3 binding. Furthermore, KCTD proteins form homotetramers and homopentamers via BTB oligomerization rather than the previously described homodimers. Despite these differences, many KCTD proteins interact with Cul3 with dissociation constants of approximately 50 nM. While the target substrates for many of the KCTD/Cul3 E3 ligase complexes are unknown, recent studies have implicated the GABAβ2 receptor as an interactor of KCTD 8, 12, 12b and 16. Here, we report the pentameric crystal structure of the KCTD9 BTB domain and our progress on the structural characterization of Cul3/KCTD/substrate complexes.