P53-binding Domain Research Articles

Analysis of the Interaction of UBE2Q1 with B4GALT1 and P53: Experimental and Molecular Modeling Study.

UBE2Q1-dependent ubiquitination of key proteins including β 1,4- galactosyltransferase (GalT1), and P53 might play a pivotal role in cancer development. The present study aimed to evaluate the molecular analysis of possible interactions between UBE2Q1 with B4GALT1 and P53 proteins. We established SW1116 colorectal cancer cell line stably transfected with UBE2Q1. To verify the overexpression of UBE2Q1, we performed western blot and fluorescent microscopy analysis. Using the immunoprecipitation (IP) product of the over-expressed protein on the silver staining gel, we observed the potential interacting partners of UBE2Q1. The Molecular Operating Environment (MOE) software was also used to perform the molecular docking of the UBC domain of UBE2Q1 (2QGX) with B4GALT1 (2AGD), and P53 (tetramerization (1AIE) and DNA binding domains (1GZH)) proteins. Western blot and IP analysis detected a UBE2Q1-GFP band in transfected cells, while no band was detected for mock-transfected cells. Moreover, the overexpression of UBE2Q1 tagged with GFP was observed under fluorescent microscopy as well with about 60-70% shining. Silver staining of IP gel revealed several bands in colorectal cancer (CRC) with UBE2Q1 overexpression. Protein- Protein interaction (PPI) analysis also depicted a high affinity of the UBC domain of UBE2Q1 to the B4GALT1 and P53 (tetramerization and DNA binding domains). Molecular docking also revealed hot-spot regions for all poses. Our data suggest that UBE2Q1 as an E2 enzyme of ubiquitination system can interact with B4GALT1 and P53, and may contribute to the accumulation of misfolded important proteins and colorectal tumor development.

Expression, purification, and characterisation of the p53 binding domain of Retinoblastoma binding protein 6 (RBBP6).

RBBP6 is a 250 kDa eukaryotic protein known to be a negative regulator of p53 and essential for embryonic development. Furthermore, RBBP6 is a critical element in carcinogenesis and has been identified as a potential biomarker for certain cancers. RBBP6's ability to interact with p53 and cause its degradation makes it a potential drug target in cancer therapy. Therefore, a better understating of the p53 binding domain of RBBP6 is needed. This study presents a three-part purification protocol for the polyhistidine-tagged p53 binding domain of RBBP6, expressed in Escherichia coli bacterial cells. The purified recombinant domain was shown to have structure and is functional as it could bind endogenous p53. We characterized it using clear native PAGE and far-UV CD and found that it exists in a single form, most likely monomer. We predict that its secondary structure is predominantly random coil with 19% alpha-helices, 9% beta-strand and 14% turns. When we exposed the recombinant domain to increasing temperature or known denaturants, our investigation suggested that the domain undergoes relatively small structural changes, especially with increased temperature. Moreover, we notice a high percentage recovery after returning the domain close to starting conditions. The outcome of this study is a pure, stable, and functional recombinant RBBP6-p53BD that is primarily intrinsically disordered.

Germline Loss-of-Function Mutations in MDM4 Cause p53-Dependent Hematopoietic Cell Death in Patients with Variable Bone Marrow Failure Phenotypes

The mouse double minute 4 (MDM4) negatively regulates p53 through the p53/MDM2/MDM4 regulatory network and is commonly overexpressed in several cancers. Here, we describe two unrelated pedigrees with germline MDM4 mutations with clinical features of bone marrow (BM) failure. Patient 1 (P1) presented at the age of 11 months with chronic pure red cell aplasia requiring chronic red blood cell transfusions and BM findings with absent erythroid precursors and dysplastic megakaryocytes. Patient 2 (P2) presented at age of 4 weeks with neutropenia with hypogranulation of neutrophils and a normocellular bone marrow with hypoplastic myelopoiesis and dysplasia of all three lineages. Both patients had normal chromosomal breakage and normal for age telomere length in peripheral blood. Whole genome and panel sequencing identified germline heterozygous variants in MDM4: p.Leu31Thrfs*15 in P1 and p.T454M in P2 as the only relevant candidate variants. Both mutations are absent in >140 thousand individuals in gnomAD database, and the constraint analysis indicates that MDM4 gene is intolerant to missense variation (observed to expected (O/E) ratio of 181/258, Z-score 1.71) and highly intolerant to protein truncating variation (O/E: 0/27 with a pLI score of 1). MDM4 protein consists of four key domains: p53 binding N-terminus, an acidic region, a zinc finger domain, and a C-terminal RING domain, which heterodimerizes with MDM2 to ubiquitinate and promote p53 degradation. MDM4 p.T454M missense mutation found in P2 was previously reported by Toufektchan et al. in 2020 in one patient with BM failure phenotype with short telomeres who carried additional TERT variant. The MDM4 p.T454M mutation was shown to result in lower protein levels (consistent with a loss-of-function effect) and had altered capacity to dimerize with the MDM2 RING domain, causing p53 hyperactivity. MDM4 p.Leu31Thrfs*15 mutation described in P1 is localized in the N-terminal p53 binding domain and results in RNA transcript loss, as confirmed by us in patient cells using RNA sequencing showing a two-fold reduction of the dominant transcript [ENST00000367182.8]. This was corroborated by Western blot demonstrating decreased levels of MDM4 protein. We sought to elucidate the impact of p.Leu31Thrfs*15 and p.T454M mutations on hematopoiesis and regulation of p53 expression. In two human induced pluripotent stem cell (iPSC) lines derived from male and female healthy donors, we knocked-in the MDM4 p.T454M and p.Leu31Thrfs*15 mutations by CRISPR/Cas9 engineering. Of note, MDM4 p.Leu31Thrfs*15 homozygous knock-in line could not be established despite multiple attempts. We discovered statistically significant decreased erythroid and myeloid differentiation capacity from MDM4-mutant iPSCs compared to isogenic control iPSCs (p <0.05). There were statistically significantly more AnnexinV+/DAPI+ mutant iPSC-derived myeloid and erythroid progenitors compared to isogenic control-derived progenitors (p <0.05), pointing to a higher rate of cell death in MDM4-mutant cells. We further demonstrated increased expression of p53 and p21 proteins in MDM4 p.Leu31Thrfs*15 iPSC clones. To better understand the loss-of-function effect of MDM4, we knocked out MDM4 in healthy CD34+ cells using CRISPR/Cas9 editing. Knock out of MDM4 with an efficiency of >80% was achieved in multiple biological replicates, resulting in increased p53 activity and fewer blast-forming erythroid (BFU-E) and myeloid colonies compared to control cells that were Cas9-edited at the AAVS1 safe harbor locus. This impairment was rescued by disruption of TP53 gene. Sequencing of individual BFU-E colonies did not reveal any cells with biallelic loss of MDM4, while similar percentages of unedited colonies and heterozygous disrupted MDM4 colonies were detected. Xenotransplantation studies using immunodeficient NBSGW mice are currently underway to explore the impact of MDM4-edited CD34+ cells in vivo. In summary, we describe two patients with germline loss-of-function MDM4 mutations that cause p53 hyperactivity which causes the unifying feature of hematopoietic defect across a BMF disorder spectrum without telomere shortening. Sharma and Bhoopalan are equal contributors

1H, 15N and 13C backbone resonance assignments of the acidic domain of the human MDM2 protein.

The human MDM2 protein regulates the tumor suppressor protein p53 by restricting its transcriptional activity and by promoting p53 degradation. MDM2 is ubiquitously expressed, with its overexpression implicated in many forms of cancer. The inhibitory effects of MDM2 on p53 have been shown to involve its N-terminal p53-binding domain and its C-terminal RING domain. The presence of an intact central acidic domain of MDM2 has also been shown to regulate p53 ubiquitination, with this domain shown to directly interact with the p53 DNA-binding domain to regulate the DNA binding activity of p53. To date, little structural information has been obtained for the MDM2 acidic domain. Thus, to gain insight into the structure and function relationship of this region, we have applied solution-state NMR spectroscopy to characterize the segment of MDM2 spanning residues 215-300. These boundaries for the acidic domain were determined on the basis of consensus observed in multiple sequence alignment. Here, we report the 1H, 15N and 13C backbone and 13Cβ chemical shift assignments and steady-state {1H}-15N heteronuclear NOE enhancement factors as a function of residue for the acidic domain of MDM2. We show that this domain exhibits the hallmarks of being a disordered protein, on the basis both of assigned chemical shifts and residue-level backbone dynamics, with localized variation in secondary structure propensity inferred from chemical shift analysis.

The MDMX Acidic Domain Uses Allovalency to Bind Both p53 and MDMX

Autoinhibition of p53 binding to MDMX requires two short-linear motifs (SLiMs) containing adjacent tryptophan (WW) and tryptophan-phenylalanine (WF) residues. NMR spectroscopy was used to show the WW and WF motifs directly compete for the p53 binding site on MDMX and circular dichroism spectroscopy was used to show the WW motif becomes helical when it is bound to the p53 binding domain (p53BD) of MDMX. Binding studies using isothermal titration calorimetry showed the WW motif is a stronger inhibitor of p53 binding than the WF motif when they are both tethered to p53BD by the natural disordered linker. We also investigated how the WW and WF motifs interact with the DNA binding domain (DBD) of p53. Both motifs bind independently to similar sites on DBD that overlap the DNA binding site. Taken together our work defines a model for complex formation between MDMX and p53 where a pair of disordered SLiMs bind overlapping sites on both proteins.

Small molecule MMRi62 targets MDM4 for degradation and induces leukemic cell apoptosis regardless of p53 status.

MDM2 and MDM4 proteins are key negative regulators of tumor suppressor p53. MDM2 and MDM4 interact via their RING domains and form a heterodimer polyubiquitin E3 ligase essential for p53 degradation. MDM4 also forms heterodimer E3 ligases with MDM2 isoforms that lack p53-binding domains, which regulate p53 and MDM4 stability. We are working to identify small-molecule inhibitors targeting the RING domain of MDM2-MDM4 (MMRi) that can inactivate the total oncogenic activity of MDM2-MDM4 heterodimers. Here, we describe the identification and characterization of MMRi62 as an MDM4-degrader and apoptosis inducer in leukemia cells. Biochemically, in our experiments, MMRi62 bound to preformed RING domain heterodimers altered the substrate preference toward MDM4 ubiquitination and promoted MDM2-dependent MDM4 degradation in cells. This MDM4-degrader activity of MMRi62 was found to be associated with potent apoptosis induction in leukemia cells. Interestingly, MMRi62 effectively induced apoptosis in p53 mutant, multidrug-resistant leukemia cells and patient samples in addition to p53 wild-type cells. In contrast, MMRi67 as a RING heterodimer disruptor and an enzymatic inhibitor of the MDM2-MDM4 E3 complex lacked MDM4-degrader activity and failed to induce apoptosis in these cells. In summary, this study identifies MMRi62 as a novel MDM2-MDM4-targeting agent and suggests that small molecules capable of promoting MDM4 degradation may be a viable new approach to killing leukemia cells bearing non-functional p53 by apoptosis.

Structural studies of antitumor compounds that target the RING domain of MDM2.

Mouse double minute 2 homolog (MDM2) is an E3 ubiquitin-protein ligase that is involved in the transfer of ubiquitin to p53 and other protein substrates. The expression of MDM2 is elevated in cancer cells and inhibitors of MDM2 showed potent anticancer activities. Many inhibitors target the p53 binding domain of MDM2. However, inhibitors such as Inulanolide A and MA242 are found to bind the RING domain of MDM2 to block ubiquitin transfer. In this report, crystal structures of MDM2 RING domain in complex with Inulanolide A and MA242 were solved. These inhibitors primarily bind in a hydrophobic site centered at the sidechain of Tyr489 at the C-terminus of MDM2 RING domain. The C-terminus of MDM2 RING domain, especially residue Tyr489, is required for ubiquitin discharge induced by MDM2. The binding of these inhibitors at Tyr489 may interrupt interactions between the MDM2 RING domain and the E2-Ubiquitin complex to inhibit ubiquitin transfer, regardless of what the substrate is. Our results suggest a new mechanism of inhibition of MDM2 E3 activity for a broad spectrum of substrates.

1H, 15N and 13C backbone resonance assignments of the acidic domain of the human MDMX protein.

The human MDMX protein, also known as MDM4, plays a pivotal role in regulating the activity of the tumor suppressor protein p53 by restricting p53 transcriptional activity and stimulating the E3 ubiquitin ligase activity of another key regulatory protein, MDM2, to promote p53 degradation. MDMX is ubiquitously expressed in most tissue types and overexpression of MDMX has been implicated in many forms of cancer. MDMX has been shown to require an intact N-terminal p53-binding domain and C-terminal RING domain to exert inhibitory effects on p53. The presence of a tryptophan-rich sequence in the central acidic domain of MDMX has also been implicated in regulating the interaction between MDMX and p53, directly interacting with the p53 DNA-binding domain. To date, little structural information has been obtained for this acidic region of MDMX that encompasses the Trp-rich sequence. In order to gain insight into the structure and function of this region, we have carried out solution-state NMR spectroscopy studies utilizing the segment of MDMX spanning residues 181-300-with bounds specifically chosen through multiple sequence alignment-which encompasses nearly 25% of MDMX. Here, we report the 1H, 15N and 13C backbone chemical shift assignments of the acidic domain of MDMX and show that it exhibits hallmarks of intrinsic disorder and localized variation in inferred secondary structure propensity.

ASPP2κ Is Expressed In Human Colorectal Carcinoma And Promotes Chemotherapy Resistance And Tumorigenesis.

Alternative splicing is a common physiologic mechanism to generate numerous distinct gene products from one gene locus, which can result in unique gene products with differing important functional outcomes depending on cell context. Aberrant alternative splicing is a hallmark of cancer that can contribute to oncogenesis and aggressiveness of the disease as well as resistance to therapy. However, aberrant splicing might also result in novel targets for cancer therapy. ASPP2 is a haplo-insufficient tumor suppressor, that functions through both p53-dependent as well as p53-independent mechanisms to enhance cell death after stress. Interestingly, the common human tumor TP53 mutations result in a loss of the binding sites to ASPP2, leading to impaired induction of apoptosis. Vice versa, attenuation of ASPP2 has been described to be associated with high-risk disease, therapy failure and poor clinical outcome especially in tumors harboring the TP53 wildtype (WT) isoform. We have recently identified a novel, dominant-negative splicing variant of ASPP2, named ASPP2κ, with oncogenic potential. Exon-skipping results in a reading-frame shift with a premature translation stop, omitting most of the ASPP2 C-terminus - which harbors the p53-binding domain. Consequently, the ASPP2-p53 interaction is abrogated, which in part impacts on oncogenesis, aggressiveness of disease and response to therapy. Since ASPP2κ has been shown in hematologic malignancies to promote tumorigenesis, we further wished to determine if aberrant ASPP2κ expression plays a role in human solid tumors. In this report, we find that ASPP2κ is frequently expressed in human colorectal tumors (CRC). Using ASPP2κ overexpressing and interference CRC models, we demonstrate a functional role of ASPP2κ in contributing to oncogenesis and resistance to therapy in CRC by 1) enhancing proliferation, 2) promoting cell migration and, 3) conferring resistance to chemotherapy induced apoptosis. Our findings have far-reaching consequences for future diagnostic and therapeutic strategies for ASPP2κ expressing colorectal cancer patients and provide proof-of-principle to further explore ASPP2κ as potential predictive marker and target for therapy in clinical trials.

Understanding the Role of Flavonoid Based Small Molecules in Modulating the Oncogenic Protein-Protein Interactions: A Quest for Therapeutic Arsenal

We explored the anticancer activity of two synthetic flavonoid-based small molecules, HMDC and HMDF, with bioactive methylenedioxy functionality. HMDF inhibited the proliferation of the p53 wild-type (NCIH460 and A549), and p53 null (NCIH1299) non-small cell lung cancer and breast cancer (MCF-7) cells more potently than HMDC without significant cytotoxic effects on the normal lung-epithelial (L132) and macrophage (Raw 264.7) cells. HMDF mediated reduction of the cell proliferation occurred due to its attachment at the p53-binding domain of MDM2 (also evident from molecular docking analysis), which induced the disruption of the p53-MDM2 interactions. Ultimately, a higher expression of p53 in the NCIH460 cells was observed. The up-regulated p53 level instigated apoptosis of cancer cells. However, MDM2 expression level remained unaltered. The docking studies further indicate that HMDF can suppress the anti-apoptotic activity of Bcl-2 protein by blocking its BH3 domain.

Tau Protein as a Fusion Tag for Recombinant Peptide/Protein Expression

The microtubule-associated protein tau plays an important role in regulating the assembly and spatial organization of microtubule in the central nervous system. Although hyperphosphorylated tau forms amyloid filaments in a number of neurodegenerative diseases known as tauopathies, such as Alzheimer's disease, unmodified tau is stable and resistant to aggregation even in boiling water. Tau protein is highly expressed in E. coli and can be easily purified from cell lysis by utilizing its thermo stability. So for, widely used tags for recombinant protein expression are folded proteins, such as glutathione S-transferase, maltose binding protein, and ubiquitin. In this work, we designed a novel recombinant protein/peptide expression system based on the microtubule-binding domain of intrinsically disordered protein tau. A His-tag was arranged at the N-terminal of tau protein to facilitate affinity purification. A TEV protease cleavage site was introduced between tau and target protein/peptide, enabling release of target protein/peptide from the tagged protein (Figure 1). To demonstrate the usability of this tau fusion expression system, we tested the expression of three proteins/peptides, i.e., the N-terminal p53 binding domain of MdmX, the azurin derived anticancer peptide p28, and the C-terminal transactivation domain of HIF-1α. Compared to GST-fused MdmX, the expression level and solubility of tau-fused MdmX was higher under identical expression temperature and IPTG concentration. p28 and HIF-1a are disordered peptides with 28 and 51 residues, respectively. Both tau-fused p28 and tau-fused HIF-1α were mainly expressed as soluble proteins at high level. Furthermore, most bacterial proteins were separated from tau-fused p28 and tau-fused HIF-1a by boiling the cell lyses. Finally, the tau-fused MdmX, p28, and HIF-1α could be efficiently purified by using Ni-NTA and the purity was higher than 90% based on SDS-PAGE (Figure 2). Therefore, the highly expressed and thermo stable disordered protein tau is expected to be applicable to prepare various recombinant proteins.

Bibenzyl analogue DS-1 inhibits MDM2-mediated p53 degradation and sensitizes apoptosis in lung cancer cells

BackgroundLung cancer is a leading fatal malignancy due to the high incidence of treatment failure. Dysfunction of the tumor suppressor p53 contributes to cancer initiation, progression, and therapeutic resistance. Targeting MDM2, a negative regulator of p53, has recently attracted interest in cancer drug research as it may restore tumor suppressive function. PurposeThe present study aimed to investigate the effect of 3,4-dihydroxy-5,4′-dimethoxybibenzyl (DS-1) on targeting MDM2 and restoring p53 function in lung cancer cells. MethodsThe efficacy of DS-1 alone or in combination with cisplatin in lung cancer cells was determined by MTT, nuclear staining, and annexin V/PI assay. The expression of apoptosis-related proteins was determined by western blot analysis. To evaluate the role of DS-1 on the stabilization and degradation of p53, cycloheximide chasing assay and immunoprecipitation were conducted, and the active form of p53 was investigated by immunofluorescent staining assay. To confirm and demonstrate the site interaction between DS-1 and the MDM2 protein, in silico computational analysis was performed. ResultsDS-1 exhibited a cytotoxic effect and sensitized lung cancer cells to cisplatin-induced apoptosis. DS-1 caused a significant increase in the cellular level of p53 protein, while the active form of p53 (phosphorylation at Ser15) was unaltered. DS-1 treatment in combination with cisplatin could enhance activated p-p53 (Ser15) and p53 downstream signaling (Bax, Bcl-2, and Akt), leading to a higher level of apoptosis. Immunoprecipitation analysis revealed that DS-1 decreased the p53-ubiquitin complex, a prerequisite step in p53 proteasomal degradation. Molecular docking simulation further evidenced that DS-1 interacts with MDM2 within the p53-binding domain by carbon–hydrogen bond interaction at Lys27, π–alkyl interactions at Ile37 and Leu30, and van der Waals interactions at Ile75, Val51, Val69, Phe67, Met38, Tyr43, Gly34, and Phe31. Treatment by DS-1 and cisplatin in patient-derivated primary lung cancer cells showed consistent effects by increasing cisplatin sensitivity. ConclusionsOur findings provide evidence that DS-1 is an MDM2 inhibitor and its underlying mechanism involves MDM2 binding and p53 induction, which may benefit the development of this compound for lung cancer treatment.

Identification of a Catalytic Active but Non-Aggregating MDM2 RING Domain Variant

As a key regulator of the tumour suppressor protein p53, MDM2 is involved in various types of cancer and has thus been an attractive drug target. So far, small molecule design has primarily focussed on the N-terminal p53-binding domain although on-target toxicity effects have been reported. Targeting the catalytic RING domain of MDM2 resembles an alternative approach to drug MDM2 with the idea to prevent MDM2-mediated ubiquitination of p53 while retaining MDM2′s ability to bind p53. The design of RING inhibitors has been limited by the extensive aggregation tendency of the RING domain, making it challenging to undertake co-crystallization attempts with potential inhibitors. Here we compare the purification profiles of the MDM2 RING domain from several species and show that the MDM2 RING domain of other species than human is much less prone to aggregate although the overall structure of the RING domain is conserved. Through sequence comparison and mutagenesis analyses, we identify a single point mutation, G443T, which greatly enhances the dimeric fraction of human MDM2 RING domain during purification. Neither does the mutation alter the structure of the RING domain, nor does it affect E2(UbcH5B)–Ub binding and activity. Hence, MDM2-G443T facilitates studies involving binding partners that would be hampered by the low solubility of the wild-type RING domain. Furthermore, it will be valuable for the development of MDM2 RING inhibitors.

Novel 1,2,4-triazole derivatives as apoptotic inducers targeting p53: Synthesis and antiproliferative activity

A series of novel thiazolo[3,2-b][1,2,4]-triazoles 3a-n has been synthesized and evaluated in vitro as potential antiproliferative. Compounds 3b-d exhibited significant antiproliferative activity. Compound 3b was the most potent with Mean GI50 1.37 µM comparing to doxorubicin (GI50 1.13 µM). The transcription effects of 3b, 3c and 3d on the p53 were assessed and compared with the reference doxorubicin. The results revealed an increase of 15–27 in p53 level compared to the test cells and that p53 protein level of 3b, 3c and 3d was significantly inductive (1419, 571 and 787 pg/mL, respectively) in relation to doxorubicin (1263 pg/mL). The docking study of the new compounds 3a-n revealed high binding scores for the new compounds toward p53 binding domain in MDM2. The docking analyses revealed the highest affinities for compounds 3b-d which induced p53 activity in MCF-7 cancer cells. Compound 3b which exhibited the highest antiproliferative activity and induced the highest increase in p53 level in MCF-7 cells showed also the highest affinity to MDM2.

Autism-Associated Chromatin Remodeler CHD8 Governs the Survival and Differentiation of Hematopoietic Stem/Progenitor Cells

Hematopoiesis is a strictly regulated process which depends on regulated proliferation, differentiation, and survival of hematopoietic stem/progenitor cells (HSPCs). Multiple signaling pathways, transcription factors and epigenetic machineries are involved in this important process. Increasing evidence from recent studies indicates that ATP-dependent chromatin-remodeling genes are involved in the control of crucial gene-expression programs in stem/progenitor cell regulation. Among them, the CHD8 gene encodes a member of chromodomain helicase DNA-binding (CHD) family of SNF2H-like ATP-dependent chromatin remodeler, mutations of which define a subtype of autism spectrum disorders. Whether CHD8 plays a role in hematopoietic cells remains unknown. In this study, to define the role of CHD8 in hematopoiesis and HSPC regulation, we carried out conditional deletion of CHD8 in the hematopoietic lineages using an interferon inducible Mx1-Cre; Chd8F/Fmouse model. As early as one week after polyI:C injection, the CHD8 knockout mice experienced drastic pancytopenia and bone marrow failure with a reduction of bone marrow (BM) cellularity to ~1/3 of that of WT controls. In the HSPC compartment, loss of CHD8 resulted in a depletion of Lin-c-Kit+ (LK) and Lin-Sca1+c-Kit+ (LSK) progenitors and a severe reduction of the GMP, MEP, MPP and CD150+CD48-LSK HSC populations. Accompanying the loss of HSPCs were dramatically increased apoptosis rate and cell cycle arrest in Chd8-/- cells. An examination of HSPC function by transplantation found that the BM cells from Chd8-/- mice could not engraft in the recipient mice that died ~20 days post-transplantation. The chimerism of Chd8-/- BM cells also reduced drastically in a competitive BM transplantation assay when induction of Chd8 deletion occurred after Mx1-Cre; Chd8F/Fcells were pre-engrafted at 1:1 ratio with WT BM cells. These results indicate that CHD8 is essential for hematopoiesis and HSPC survival and proliferation. To elucidate the underlying mechanism of CHD8 function in HSPCs, we performed complementary biochemical, genomic and genetic analyses of the WT (Chd8F/F) and Chd8-/-HSPC cells. Firstly, we found blood progenitor cells dominantly express the short form of CHD8 containing residues 1 to 751 which retains the N-terminal P53 binding domain, rather than the long form of the CHD8 protein. Secondly, we revealed by RNA-seq and subsequent RT-PCR analyses that the P53 signaling pathway signatures including P53, P21, Bax and Noxa are aberrantly activated in Chd8-/-HSPCs. At the protein level, P53 and P21 are significantly elevated in Chd8-/- LK cells. Thirdly, ATAC-seq analysis of the LSK cells revealed increased global chromatin accessibilities after CHD8 deletion, including the promoter regions of P21 and Noxa genes. By a "Cut &amp; Run" assay using H3K4me3 antibody in Chd8F/Fand Chd8-/- LSK cells we have also seen significantly enhanced epigenetic H3K4me3 mark on the promoter region of P21 and Noxa. An anti-CHD8 "Cut &amp; Run" further detected direct binding sites of CHD8 on P53 and P21, but not Noxa or Bax gene. Fourthly, we observed a direct binding interaction by co-immunoprecipitation between P53 and CHD8 proteins. Finally, genetic deletion of one allele of P53 gene in Chd8-/- HSPCs (P53+/-;Chd8-/-) was able to completely suppress the Bax/Noxa expression and apoptosis phenotype and rescue most of the HSC and early progenitor defects but not the later stage of differentiation defects and BM cellularity loss. Interestingly, deletion of both alleles of P53 gene (P53-/-;Chd8-/-) was able to further remove the differentiation block between LSK and LK cells observed in the P53+/-;Chd8-/-background which is associated with a P21 mediated G0 cell cycle phase arrest. These combined results suggest a mechanism of action that CHD8 works through a direct complex with P53 to regulate P21 gene expression which mediates cell cycle control of HSPC differentiation and an indirect CHD8 mediated P53 expression and subsequent P53 regulated Noxa/Bax expression which regulates HSPC survival. Thus, we identify the autism-associated CHD8 as an essential chromatin suppressor in regulating hematopoiesis through restricting the expression of P53 and chromatin accessibility of P53 downstream targets, Bax/Noxa and P21, to enable selective control of HSPC survival and differentiation, respectively. Disclosures No relevant conflicts of interest to declare.

Expression Analysis of RbBP6 in human cancers: a Prospective biomarker.

Retinoblastoma binding protein 6 (RBBP6) is a cancer-related protein that has been implicated in the regulation of cell cycle and apoptosis. RBBP6 isoform 1 has been demonstrated to interact with two tumour suppressors, p53 and pRB. Isoform 1 been shown to regulate p53 through its ubiquitin ligase activity, thus implicating in cell cycle regulation and apoptosis. Isoforms 1 and 2 are multidomain proteins containing a domain with no name (DWNN) domain, a Zinc Finger, a RING Finger, an Rb-binding domain and a p53-binding domain. The RBBP6 isoform 3 comprises the DWNN domain only. Isoform 4 lacks the Rb-binding domain but its role is less understood. RBBP6 isoform 3 has been reported as a cell cycle regulator with anticancer potential. There have been several studies that have clearly demonstrated that RBBP6 may be an important biomarker for cancer diagnosis and a potential drug target for cancer treatment. This work focused on differential expression of RBBP6 transcripts in different cancers, providing detailed analysis of their potential as diagnostic biomarkers for different cancers. These cancers include breast, liver, cervical and colon carcinomas. The expression of RBBP6 transcripts may further provide better understanding of the role of the RBBP6 in carcinogenesis and cell homeostasis.

Abstract 3040: The expressional effects of RBBP6 in breast cancer cells are p53-dependent

Abstract The incidence rate of breast cancer has increased beyond that of lung cancer, making it the most common malignancy among women. Breast tumor progression is partly because of p53 inactivation by overexpressed that possess p53 binding domain. RBBP6 forms a member of ubiquitous regulatory proteins because its RING finger-like domain has an E3 ligase activity. The overexpression of RBBP6 in several malignancies makes it a potential target in cancer management. However, there is no evidence on whether the effect of RBBP6 on cell growth and apoptosis is cell line dependent, more especially in breast cancer cell lines that have distinct p53 expression profiles. Therefore, the aim of this study was to evaluate the RBBP6 effects on cell growth and apoptosis in breast cancer cell lines with different p53 expression profiles. RBBP6 expression was successfully manipulated using gene silencing and protein overexpression techniques in MCF-7 and MDA-MB-231 cell lines. Transfected cells were co-treated with anti-cancer agents followed by apoptosis detection using confocal microscopy and flow cytometry, which was further confirmed by caspase 3/7 activity and quantification of apoptotic genes. RBBP6 was overexpressed in breast cancer tissues that were classified as stage 3 and 4 while in stage 1 it was expressed but at much lower levels. The wt. p53-expressing MCF-7 cell line was more susceptible to apoptosis induction as opposed to the mt. p53-expressing MDA-MB-231. RBBP6 silencing led to a significant accumulation of p53 expression in MCF-7 as compared to MDA-MB-231. Co-treatment with GABA and camptothecin seemed to sensitize the cells to apoptosis induction. These data suggest that RBBP6 silencing triggers significant levels of intrinsic apoptosis and over-expression appears to promote cell proliferation in wild-type p53-expressing MCF-7 rather than in MDA-MB-231 cells. In conclusion, the effect of RBBP6 on cell proliferation and apoptosis induction in breast cancer seem to be cell line dependent based on p53 status. Note: This abstract was not presented at the meeting. Citation Format: Pontsho Moela, Lesetja Motadi, Marcia Lekganyane. The expressional effects of RBBP6 in breast cancer cells are p53-dependent [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3040.

Abstract 4506: MDM2 alternative splicing is regulated by microRNA binding to control p53 activity

Abstract Alternative splicing of the MDM2 is an important means by which p53 is upregulated to combat the deleterious effects of genotoxic stress. One splice variant, MDM2-ALT1, is activated in response to genotoxic stress and is comprised of only the two terminal coding exons 3 and 12 and therefore lacks a p53 binding domain. This variant has been identified in a number of human tumors, including invasive carcinoma of the breast and lung, as well as soft tissue sarcomas. Despite its pervasiveness in tumors and the therapeutic possibilities it presents, there is very little known about the regulators of that govern this alternative splicing event. Our lab has identified a specific microRNA that is expressed at high levels in normal cells but decreased under conditions of genotoxic stress and in rhabdomyosarcoma cells, both of which are characterized by the expression of the MDM2 spliced isoform. Hence, expression of the microRNA is inversely correlated with the splice variant MDM2-ALT1. Though recent studies have shown that splicing factors can be regulated through miRNA-mediated gene silencing and indirectly influence splicing choices, we were unable to identify any splicing regulators of MDM2 to be targeted by the microRNA. Interestingly, the microRNA has been reported as one of the miRNAs that is reimported to the nucleus but has not been ascribed a role in the nuclear space. We therefore wanted to test the hypothesis that the microRNA can directly bind to the MDM2 pre-mRNA to drive splicing choices in a regulated fashion. To this end, we have identified predicted binding sites for the microRNA in the regulated region of MDM2, within and near exons 3 and 12. We have shown that the microRNA binds specifically to these regions in MDM2 in normal cells and has decreased binding after genotoxic stress. Furthermore, we have shown that overexpression of the pre-microRNA can alleviate the induction of the MDM2-ALT1 isoform in response to genotoxic stress. We propose that microRNA binding to the MDM2 pre-mRNA regulates its splicing and plays a role in the DNA damage response and the pathway to tumorigenesis. Our work implicates for the first time that a miRNA would be acting as a splicing factor, a novel function for this class of non-coding RNAs and a novel method of regulation of the p53-MDM2 axis. Furthermore, our discoveries reveal miR-regulated splicing as a possible strategy to activate p53 and sensitize cancer cells to conventional cancer therapies. Citation Format: Matias Montes, Andrew Goodwin, Dawn S. Chandler. MDM2 alternative splicing is regulated by microRNA binding to control p53 activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4506.

Recombinant Butelase-Mediated Cyclization of the p53-Binding Domain of the Oncoprotein MdmX-Stabilized Protein Conformation as a Promising Model for Structural Investigation

Cyclization of the polypeptide backbone has proven to be a powerful strategy for enhancing protein stability for fundamental research and pharmaceutical application. The use of such an approach is restricted by how well a targeted polypeptide can be efficiently ligated. Recently, an Asx-specific peptide ligase identified from a tropical cyclotide-producing plant and named butelase 1 exhibited excellent cyclization kinetics that cannot be matched by other known ligases, including intein, PATG, PCY1, and sortase A. In this work, we aimed to examine whether butelase 1 facilitated protein conformational stability for structural investigation. First, we successfully expressed recombinant butelase 1 (rBTase) in the yeast Pichia pastoris. Next, rBTase was shown to be highly efficient in the cyclization of the p53-binding domain (N-terminal domain) of murine double minute X (N-MdmX), an important target for designing anticancer drugs. The cyclized N-MdmX (cMdmX) exhibited increased conformational stability and improved interaction with the ligand compared with those of noncyclized N-MdmX. Importantly, the thermal melting process was completely reversible, contrary to noncyclized N-MdmX, and the melting temperature ( Tm) of cMdmX was increased to 47 from 43 °C. This stable conformation of cMdmX was further confirmed by 15N-1H heteronuclear single-quantum coherence nuclear magnetic resonance (NMR) spectroscopy. The complex of cMdmX and the ligand was tested for protein crystallization, and several promising findings were revealed. Therefore, our work not only provides a recombinant version of butelase 1 but also suggests a conventional approach for preparing stable protein samples for both protein crystallization and NMR structural investigation.

RBBP6 expressional effects on cell proliferation and apoptosis in breast cancer cell lines with distinct p53 statuses.

IntroductionBreast cancer is the most common malignancy amongst women and has a higher incidence rate than lung cancer. Its tumor progression partially results from inactivation of p53 which is caused by overexpression of ubiquitous regulatory proteins possessing p53-binding domain. RBBP6 is regarded as one of the ubiquitous proteins because of its RING finger-like domain which enables it to possess E3 ligase activity. Thus, it has become a potential target in cancer treatment as it is highly expressed in various malignancies including cancer. However, it is not clearly defined whether the effect of RBBP6 on cell growth and apoptosis is cell line-dependent, more especially in breast cancer cell lines that have distinct p53 expression profiles. This study aims at evaluating the effects of RBBP6 on cell growth and apoptosis in breast cancer cell lines with different p53 expressions.MethodsFollowing the analysis at mRNA and protein levels in breast cancer tissue, RBBP6 expression was successfully manipulated using gene silencing and protein overexpression techniques in MCF-7 and MDA-MB-231 cell lines. The cells were co-treated with siRBBP6 and anticancer agents following apoptosis detection, which was confirmed by caspase 3/7 activity and quantification of apoptotic genes.ResultsRBBP6 was overexpressed in breast cancer tissues that were classified as stages 3 and 4, while in stage 1, its expression was much lower. The MCF-7 cell line which expresses wild-type p53 was more sensitive to apoptosis induction than MDA-MB-231 which is a mutant p53-expressing cell line. These data suggest that RBBP6 silencing triggers significant levels of intrinsic apoptosis, and its overexpression appears to promote cell proliferation in wild-type p53-expressing MCF-7 cell line as opposed to MDA-MB-231 cells.ConclusionThe effect of RBBP6 on cell proliferation and apoptosis induction in breast cancer seems to be cell line-dependent based on p53 status.