Sibling rivalry among the ZBTB transcription factor family: homo vs. heterodimers

The BTB domain is an oligomerization domain found in over 200 proteins encoded in the human genome. In the family of BTB domain and Zinc Finger-containing (ZBTB) transcription factors, 49 members share the same protein architecture. The N-terminal BTB domain is structurally conserved among the family members and serves as the dimerization site while the C-terminal zinc finger motifs mediate DNA binding. The available BTB domain structures from this family reveal a natural inclination for homodimerization. In this study we investigated the potential for heterodimer formation in the cellular environment. We selected five BTB homodimers and four heterodimer structures. We performed in vitro binding assays with fluorescent protein-BTB domain fusions to assess dimer formation. We tested the binding of several BTB pairs, and we were able to confirm the heterodimeric physical interaction between the BTB domains of PATZ1 and PATZ2, previously reported only in an interactome mapping experiment. We also found this pair to be co-expressed in several immune system cell types. Finally, we used the available structures of BTB domain dimers and newly constructed models in extended molecular dynamics simulations (500 ns) to understand the energetic determinants of homo and heterodimer formation. We conclude that heterodimer formation, although frequently described as less preferred than homodimers, is a possible mechanism to increase the combinatorial specificity of this transcription factor family.

AlphaFold-Predicted Structures of KCTD Proteins Unravel Previously Undetected Relationships among the Members of the Family

One of the most striking features of KCTD proteins is their involvement in apparently unrelated yet fundamental physio-pathological processes. Unfortunately, comprehensive structure–function relationships for this protein family have been hampered by the scarcity of the structural data available. This scenario is rapidly changing due to the release of the protein three-dimensional models predicted by AlphaFold (AF). Here, we exploited the structural information contained in the AF database to gain insights into the relationships among the members of the KCTD family with the aim of facilitating the definition of the structural and molecular basis of key roles that these proteins play in many biological processes. The most important finding that emerged from this investigation is the discovery that, in addition to the BTB domain, the vast majority of these proteins also share a structurally similar domain in the C-terminal region despite the absence of general sequence similarities detectable in this region. Using this domain as reference, we generated a novel and comprehensive structure-based pseudo-phylogenetic tree that unraveled previously undetected similarities among the protein family. In particular, we generated a new clustering of the KCTD proteins that will represent a solid ground for interpreting their many functions.

Mechanisms of CP190 Interaction with Architectural Proteins in Drosophila Melanogaster

Most of the known Drosophila architectural proteins interact with an important cofactor, CP190, that contains three domains (BTB, M, and D) that are involved in protein–protein interactions. The highly conserved N-terminal CP190 BTB domain forms a stable homodimer that interacts with unstructured regions in the three best-characterized architectural proteins: dCTCF, Su(Hw), and Pita. Here, we identified two new CP190 partners, CG4730 and CG31365, that interact with the BTB domain. The CP190 BTB resembles the previously characterized human BCL6 BTB domain, which uses its hydrophobic groove to specifically associate with unstructured regions of several transcriptional repressors. Using GST pull-down and yeast two-hybrid assays, we demonstrated that mutations in the hydrophobic groove strongly affect the affinity of CP190 BTB for the architectural proteins. In the yeast two-hybrid assay, we found that architectural proteins use various mechanisms to improve the efficiency of interaction with CP190. Pita and Su(Hw) have two unstructured regions that appear to simultaneously interact with hydrophobic grooves in the BTB dimer. In dCTCF and CG31365, two adjacent regions interact simultaneously with the hydrophobic groove of the BTB and the M domain of CP190. Finally, CG4730 interacts with the BTB, M, and D domains of CP190 simultaneously. These results suggest that architectural proteins use different mechanisms to increase the efficiency of interaction with CP190.

Differential expression of ZBTB20 in cancer of the vulva.

In these brief notes we document work using published microarray data (1, 2) to pioneer integrative transcriptome analysis comparing vulvar carcinoma to its tissue of origin, the vulva. We report the differential expression of zinc finger and BTB domain-containing 20, encoded by ZBTB20, in cancer of the vulva. ZBTB20 may be of pertinence to understanding transformation and disease progression in vulvar cancer (3).

ZBTB38 in Porcine Alveolar Macrophages Positively Regulates the Proliferation of Japanese Encephalitis Virus

Background Japanese encephalitis (JE) is an important zoonotic disease caused by Japanese encephalitis virus (JEV), and pigs are intermediate host of this disease. Previous studies have confirmed that JEV can proliferate in the respiratory tract of mice and spread through it. Therefore, this study aimed to screen the proteins interacting with JEV on porcine alveolar macrophage cell and verify its role in the proliferation of JEV.Methods and results Porcine alveolar macrophages cell line 3D4/21 were infected with JEV, and obvious cytopathic effect (CPE) was observed. Zinc finger and BTB domain containing 38 (ZBTB38) was screened out as an interacting protein using co-immunoprecipitation assay and validated through knockout and overexpression of ZBTB38 in 3D4/21 cells. The results demonstrated that loss of ZBTB38 function basically had no effect on the attachment and entry processes of JEV, while the transcription level of JEV envelope gene, the expression level of NS3 protein and the number of virions were all significantly down-regulated in the subsequent infection stage. Conclusion Overall, one core conclusion was drawn in this paper that ZBTB38 promotes the proliferation of JEV especially in the middle and late stages of infection. This study provides new information for understanding the pathogenic mechanism of JEV, especially the respiratory transmission caused by JEV infection.

Abstract P239: Identification Of Binding Sites For Phosphodiesterase 5 (PDE5) On Rho-related BTB Domain Containing 1 (RhoBTB1) As A Guide To Identifying Novel RhoBTB1-Binding Partners

In-depth studies of molecular pathways have provided insights to explore therapeutic targets for treating hypertension as a complex disease. We previously identified PDE5 as an interacting partner with RhoBTB1 protein in aortic smooth muscle cells (SMCs). RhoBTB1 regulates PDE5 activity and therefore intracellular cGMP levels, and restoration of RhoBTB1 expression in RhoBTB1-deficient states results in improvement of cardiovascular status in a mouse model of hypertension. Here we identified the specific regions of RhoBTB1 that are responsible for the recruitment of PDE5. Our goal is to use this as a guide to identify additional RhoBTB1 interacting proteins which play important roles in cardiovascular function in the hope their identification may open novel therapeutic avenues against hypertension and associated pathologies. We hypothesize that RhoBTB1 acts as an adaptor for Cullin-3 E3 ring ubiquitin ligase complex, where Cullin-3 functions as “scaffold” which delivers PDE5 (and other targets) to Cullin-3 complex followed by ubiquitination and proteasomal degradation. We generated several truncations in full length RhoBTB1, splitting it into its GTPase, BTB1, BTB2, Carboxyl-terminal (CT), BTB1-BTB2, and BTB1-BTB2-CT domains. To check the preferred binding interface for PDE5, we co-transfected HEK293 cells with epitope tagged PDE5 and separately tagged truncations of RhoBTB1, and analyzed the interaction between PDE5 and RhoBTB1 domains using co-immunoprecipitation assays. Our result shows that BTB1-BTB2-C domain of RhoBTB1 is the preferred binding region for PDE5. The BTB1-BTB2 domain lacking the CT was unable to bind to PDE5. Next, we will utilize stable isotope labeling by amino acids in cell culture (SILAC) followed by proteomic profiling to identify additional targets and/or adaptors involved in the binding of PDE5 and RhoBTB1. Similarly, we will tag specific domains of RhoBTB1 using the ascorbate peroxidase (APEX2) fusion system followed by proteomic profiling.

Abstract MP47: Rho-related BTB Domain-containing Protein 1 (RhoBTB1) Attenuates Established Arterial Stiffness In Ang-ii-treated Mice

RhoBTB1 is a transcription target of PPARγ but its function remains unclear. Restoration ofRhoBTB1 by genetic complementation in S-P467L mice expressing dominant negative PPARγmutation reversed hypertension, arterial stiffness, and vascular dysfunction. Here, we asked ifrestoring RhoBTB1 can reverse Ang-II-mediated hypertension and arterial stiffness.To test this hypothesis, S-RhoBTB1 mice, which expressed smooth muscle-specific, tamoxifen(Tx)-inducible RhoBTB1, were implanted with minipumps which released Ang-II (490ng/kg/min, 6weeks) or vehicle. Concurrently, littermate ISM-Cre mice, which expressed smooth muscle-specific, Tx-inducible Cre (but not RhoBTB1), were used as control. Transgenes were induced by5 daily Tx injections 2 weeks after Ang-II pump implantation, and tissue was harvested 2 weeksafter Tx. Measurements were made during a 1 week baseline period, 2 weeks of Ang-II, and 2weeks after transgene activation.Aortic RhoBTB1 expression was decreased in Ang-II-treated ISM-Cre mice (ISM-Cre, Ang-II vs.ISM-Cre, vehicle dCT: 13.6±0.1 vs. 12.4±0.1, n=10), and restored to normal in Ang-II-treated S-RhoBTB1 mice (S-RhoBTB1, Ang-II vs. ISM-Cre, vehicle dCT: 12.8±0.2 vs. 12.4±0.1, n=10-12).Interestingly, pulse-wave velocity, a clinical indicator of arterial stiffness, was significantlydecreased upon RhoBTB1 restoration in Ang-II-treated mice (S-RhoBTB1 vs. ISM-Cre: 2.9±0.1vs. 3.8±0.2, n=22-26). Concomitantly, measures of both pressure-diameter and stress-strainshowed that aortic compliance was improved by RhoBTB1 restoration in Ang-II-treated mice.Interestingly, restoring RhoBTB1 was insufficient to attenuate Ang-II-mediated hypertension (S-RhoBTB1, Ang-II vs. ISM-Cre, Ang-II vs. ISM-Cre, vehicle SBP: 152.1±1.7 vs. 148.0±3.7 vs.115.4± 0.7 mmHg n=12, 8, 6). Currently, we are investigating the mechanism by which RhoBTB1attenuates arterial stiffness.In summary, RhoBTB1 restoration ameliorated Ang-II-mediated arterial stiffness but nothypertension, suggesting that arterial stiffness is not merely a consequence of hypertension butcan be independently regulated from blood pressure. Moreover, there may be novel targets ofRhoBTB1 that can regulate aortic stiffness and be therapeutic.

ZBTB20 is critical for the specification of a subset of callosal projection neurons and astrocytes in the mammalian neocortex

Neocortical progenitor cells generate subtypes of excitatory projection neurons in sequential order followed by the generation of astrocytes. The transcription factor Zinc Finger and BTB Domain-Containing Protein 20 (ZBTB20) has been implicated in regulating cell specification during neocortical development. Here we show that ZBTB20 instructs the generation of a subset of callosal projections neurons in cortical layers II/III. Conditional deletion of Zbtb20 in cortical progenitors, and to a lesser degree in differentiating neurons, leads to an increase in the number of layer IV neurons at the expense of layer II/III neurons. Astrogliogenesis is also affected in the mutants with an increase in the number of a specific subset of astrocytes expressing GFAP. Astrogliogenesis is more severely disrupted by a ZBTB20 protein containing dominant mutations linked to Primrose Syndrome suggesting that ZBTB20 acts in concert with other ZBTB proteins that were also affected by the dominant negative protein to instruct astrogliogenesis. Overall, our data suggest that ZBTB20 acts both in progenitors and postmitotic cells to regulate cell-fate specification in the mammalian neocortex.