Domain Protein Family Research Articles

Direct Knockdown of PDZ and LIM Domain 1 Using an Adenoviral Delivery System Accelerates Osteogenesis and Fracture Healing in Mice.

Substantial advances have been made in understanding the role of partial PDZ and LIM domain family's proteins in skeletal-related diseases. Yet, little is known about the effect of PDZ and LIM Domain 1 (Pdlim1) on osteogenesis and fracture repair. This study aimed to investigate whether direct gene delivery using an adenovirus vector carrying Pdlim1 (Ad-oePdlim1) or encoding shRNA-Pdlim1 (Ad-shPdlim1) could affect the osteogenic activity of preosteoblastic MC3T3-E1 cells in vitro, and influence the fracture healing of mice in vivo. We found that Ad-shPdlim1 transfection contributed to the calcified nodule formation in MC3T3-E1 cells. Downregulation of Pdlim1 enhanced the alkaline phosphatase activity and increased the expression of osteogenic markers (Runt-related transcription factor 2 [Runx2], collagen type I alpha 1 chain [Col1A1], osteocalcin [OCN], and osteopontin [OPN]). Further analysis indicated that Pdlim1 knockdown could activate β-catenin signaling, as evidenced by the accumulation of β-catenin in the nucleus and the increase levels of downstream regulators such as Lef1/Tcf7, axis inhibition protein 2, cyclin D1, and SRY-box transcription factor 9. By contrast, Pdlim1 overexpression resulted in inhibition of the osteogenic activity of MC3T3-E1 cells. In vivo, at day 3 after fracture,Ad-shPdlim1 adenovirus particles were injected into the fracture site of the femur of mice, and the process of fracture healing was evaluated by X-ray, micro-computed tomography and histological examination. Local injection of Ad-shPdlim1 promoted the early cartilage callus formation, restored bone mineral density, and accelerated cartilaginous ossification, with the upregulation of osteogenic gene (Runx2, Col1A1, OCN, and OPN) expression and activation of β-catenin signaling. Thus, we concluded that inhibition of Pdlim1 contributed to osteogenesis and fracture healing by activating the β-catenin signaling pathway.

The Plasmodium falciparum exported J domain proteins fine-tune human and malarial Hsp70s: pathological exploitation of proteostasis machinery.

Cellular proteostasis requires a network of molecular chaperones and co-chaperones, which facilitate the correct folding and assembly of other proteins, or the degradation of proteins misfolded beyond repair. The function of the major chaperones, heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90), is regulated by a cohort of co-chaperone proteins. The J domain protein (JDP) family is one of the most diverse co-chaperone families, playing an important role in functionalizing the Hsp70 chaperone system to form a powerful protein quality control network. The intracellular malaria parasite, Plasmodium falciparum, has evolved the capacity to invade and reboot mature human erythrocytes, turning them into a vehicles of pathology. This process appears to involve the harnessing of both the human and parasite chaperone machineries. It is well known that malaria parasite-infected erythrocytes are highly enriched in functional human Hsp70 (HsHsp70) and Hsp90 (HsHsp90), while recent proteomics studies have provided evidence that human JDPs (HsJDPs) may also be enriched, but at lower levels. Interestingly, P. falciparum JDPs (PfJDPs) are the most prominent and diverse family of proteins exported into the infected erythrocyte cytosol. We hypothesize that the exported PfJPDs may be an evolutionary consequence of the need to boost chaperone power for specific protein folding pathways that enable both survival and pathogenesis of the malaria parasite. The evidence suggests that there is an intricate network of PfJDP interactions with the exported malarial Hsp70 (PfHsp70-x) and HsHsp70, which appear to be important for the trafficking of key malarial virulence factors, and the proteostasis of protein complexes of human and parasite proteins associated with pathology. This review will critically evaluate the current understanding of the role of exported PfJDPs in pathological exploitation of the proteostasis machinery by fine-tuning the chaperone properties of both human and malarial Hsp70s.

Dissecting the roles of MBD2 isoforms and domains in regulating NuRD complex function during cellular differentiation

The Nucleosome Remodeling and Deacetylation (NuRD) complex is a crucial regulator of cellular differentiation. Two members of the Methyl-CpG-binding domain (MBD) protein family, MBD2 and MBD3, are known to be integral, but mutually exclusive subunits of the NuRD complex. Several MBD2 and MBD3 isoforms are present in mammalian cells, resulting in distinct MBD-NuRD complexes. Whether these different complexes serve distinct functional activities during differentiation is not fully explored. Based on the essential role of MBD3 in lineage commitment, we systematically investigated a diverse set of MBD2 and MBD3 variants for their potential to rescue the differentiation block observed for mouse embryonic stem cells (ESCs) lacking MBD3. While MBD3 is indeed crucial for ESC differentiation to neuronal cells, it functions independently of its MBD domain. We further identify that MBD2 isoforms can replace MBD3 during lineage commitment, however with different potential. Full-length MBD2a only partially rescues the differentiation block, while MBD2b, an isoform lacking an N-terminal GR-rich repeat, fully rescues the Mbd3 KO phenotype. In case of MBD2a, we further show that removing the methylated DNA binding capacity or the GR-rich repeat enables full redundancy to MBD3, highlighting the synergistic requirements for these domains in diversifying NuRD complex function.

Expression of YT Domain Family Protein in Oral Squamous Cell Carcinoma and Its Correlation with the Prognosis.

Proteins contained in the conserved YTH521-b homologous (YTH) domain, have m6A-dependent RNA binding activity. As an important part of YTH domain family proteins, YTHDF1 and YTHDF3 were shown to be associated with many cancers. This paper aimed to reveal the relationship between the expression of these two proteins and the clinical prognosis of OSCC, providing certain guidance for clinical treatment of OSCC. We detected the expression of YTHDF1 and YTHDF3 in 120 OSCC patients by immunohistochemical analysis. Statistical analysis was used to determine whether the high or low expression of these two genes was significantly associated with age, gender, histological type, clinical stage, or lymph node metastasis. The correlation curve and survival curve of the two genes were produced to evaluate the potential clinical significance. We find the expression of YTHDF1 and YTHDF3 was increased in OSCC tissues compared to adjacent normal tissues. The statistical analysis showed that the expression of YTHDF1 and YTHDF3 was significantly associated with the clinical stage and histological type in OSCC patients. There was also a significant correlation between the expression of YTHDF1 and YTHDF3. A high expression of YTHDF1 and YTHDF3 was related to poor patient prognosis. Our findings suggest that a high expression of YTHDF1 and YTHDF3 may be related to poor patient prognosis.

Role of Gasdermin E in the Biogenesis of Apoptotic Cell-Derived Exosomes.

The gasdermins are a family of pore-forming proteins that has recently been suggested to play a central role in pyroptosis. In this study, we describe the novel roles of gasdermins in the biogenesis of apoptotic cell-derived exosomes. In apoptotic human HeLa and HEK293 cells, GSDMA, GSDMC, GSDMD, and GSDME increased the release of apoptotic exosomes. GSDMB and DFNB59, in contrast, negatively affected the release of apoptotic exosomes. GSDME at its full-length and cleaved forms was localized in the exosomes and exosomal membrane. Full-length and cleaved forms of GSDME are suggested to increase Ca2+ influx to the cytosol through endosomal pores and thus increase the biogenesis of apoptotic exosomes. In addition, the GSDME-mediated biogenesis of apoptotic exosomes depended on the ESCRT-III complex and endosomal recruitment of Ca2+-dependent proteins, that is, annexins A2 and A7, the PEF domain family proteins sorcin and grancalcin, and the Bro1 domain protein HD-PTP. Therefore, we propose that the biogenesis of apoptotic exosomes begins when gasdermin-mediated endosomal pores increase cytosolic Ca2+, continues through the recruitment of annexin-sorcin/grancalcin-HD-PTP, and is completed when the ESCRT-III complex synthesizes intraluminal vesicles in the multivesicular bodies of dying cells. Finally, we found that GSDME-bearing tumors released apoptotic exosomes to induce inflammatory responses in the invivo mouse 4T1 orthotropic model of BALB/c breast cancer. The data indicate that the switch from apoptosis to pyroptosis could drive the transfer of mass signals to nearby or distant living cells and tissues by way of extracellular vesicles, and that gasdermins play critical roles in that process.

Network analysis of chromophore binding site in LOV domain

Photoreceptor proteins are versatile toolbox for developing biosensors for optogenetic applications. These molecular tools get activated upon illumination of blue light, which in turn offers a non-invasive method for gaining high spatiotemporal resolution and precise control of cellular signal transduction. The Light-Oxygen-Voltage (LOV) domain family of proteins is a well-recognized system for constructing optogenetic devices. Translation of these proteins into efficient cellular sensors is possible by tuning their photochemistry lifetime. However, the bottleneck is the need for more understanding of the relationship between the protein environment and photocycle kinetics. Significantly, the effect of the local environment also modulates the electronic structure of chromophore, which perturbs the electrostatic and hydrophobic interaction within the binding site. This work highlights the critical factors hidden in the protein networks, linking with their experimental photocycle kinetics. It presents an opportunity to quantitatively examine the alternation in chromophore's equilibrium geometry and identify details which have substantial implications in designing synthetic LOV constructs with desirable photocycle efficiency.

The increase of Nrf2 m6A modification induced by FTO downregulation promotes hippocampal neuron injury and aggravates the progression of epilepsy in a rat model

Epilepsy is a common chronic neurological disorder characterized by widespread neuronal death. The purpose of this study was to investigate the role of nuclear factor erythroid 2-related factor 2 (Nrf2) m6A methylation in epilepsy. To create epileptic models, the rats were given Lithium chloride and pilocarpine, and isolated primary rat hippocampal neurons were cultured in an Mg2+ -free medium. The frequency of seizures was recorded in the epilepsy group of rats. The functional tests included TUNEL, MTT, and flow cytometry. Mechanistically, RNA degradation assay, RNA immunoprecipitation, and methylated RNA immunoprecipitation were performed. In epileptic models, Nrf2 and fat mass and obesity-associated (FTO) levels were downregulated, whereas YT521-B homology (YTH) domain family protein 2 (YTHDF2) was upregulated. Additionally, in epileptic models, there was a rise in the m6A methylation level of Nrf2 mRNA. Overexpressing FTO increased cell viability and reduced apoptosis, but Nrf2 interference reversed these effects. Meanwhile, FTO overexpression decreased the m6A methylation of Nrf2 mRNA. Moreover, YTHDF2 bound to Nrf2 mRNA and decreased its stability. Furthermore, FTO overexpression reduced seizure frequency in rats and inhibited hippocampal neuron apoptosis via lowering the m6A methylation level of Nrf2 mRNA. Overexpressing FTO reduced m6A methylation of Nrf2 mRNA, increased cell viability, suppressed apoptosis, and slowed the progression of epileptic diseases, which is linked to YTHDF2 binding to m6A-modified Nrf2 and promoting its degradation, as well as downregulating Nrf2 expression in hippocampal neurons.

Peptidoglycan recognition in Drosophila is mediated by LysMD3/4

Microbial recognition is a key step in regulating the immune signaling pathways of multicellular organisms. Peptidoglycan, a component of the bacterial cell wall, exhibits immune stimulating activity in both plants and animals. Lysin motif domain (LysMD) family proteins are ancient peptidoglycan receptors that function in bacteriophage and plants. This report focuses on defining the role of LysMD-containing proteins in animals. Here, we characterize a novel transmembrane LysMD family protein. Loss-of-function mutations at the lysMD3/4 locus in Drosophila are associated with systemic innate immune activation following challenge, so we refer to this gene as immune active (ima). We show that Ima selectively binds peptidoglycan, is enriched in cell membranes, and is necessary to regulate terminal innate immune effectors through an NF-kB-dependent pathway. Hence, Ima fulfills the key criteria of a peptidoglycan pattern recognition receptor. The human Ima ortholog, hLysMD3, exhibits similar biochemical properties. Together, these findings establish LysMD3/4 as the founding member of a novel family of animal peptidoglycan recognition proteins.

Targeted RASSF1A expression inhibits proliferation of HER2‑positive breast cancer cells invitro.

Human epidermal growth factor receptor 2-positive (HER2+) breast cancer, which accounts for 15-20% of all breast cancer, is associated with tumor recurrence and poor prognosis. RAS association domain family protein 1 subtype A (RASSF1A) is a tumor suppressor that is silenced in a variety of human cancers. The present study aimed to investigate the role of RASSF1A in HER2+ breast cancer and the therapeutic potential of RASSF1A-based targeted gene therapy for this malignancy. RASSF1A expression in human HER2+ breast cancer tissues and cell lines was evaluated by reverse transcription PCR and western blot analysis. The associations between tumorous RASSF1A level and tumor grade, TNM stage, tumor size, lymph node metastasis and five-year survival were examined. HER2+ and HER2-negative (HER2-) breast cancer cells were transfected with a lentiviral vector (LV-5HH-RASSF1A) that could express RASSF1A under the control of five copies of the hypoxia-responsive element (5HRE) and one copy of the HER2 promoter (HER2p). Cell proliferation was evaluated by the MTT and colony formation assays. It was found that tumorous RASSF1A level was negatively associated with tumor grade (P=0.014), TNM stage (P=0.0056), tumor size (P=0.014) and lymph node metastasis (P=0.029) and positively associated with five-year survival (P=0.038) in HER2+ breast cancer patients. Lentiviral transfection of HER2+ breast cancer cells resulted in increased RASSF1A expression and decreased cell proliferation, especially under hypoxic conditions. However, lentiviral transfection of HER2-breast cancer cells did not affect RASSF1A expression. In conclusion, these findings verified the clinical significance of RASSF1A as a tumor suppressor in HER2+ breast cancer and supported LV-5HH-RASSF1A as a potential targeted gene therapy for this malignancy.

Profiling of N6-methyladenosine methylation in porcine longissimus dorsi muscle and unravelling the hub gene ADIPOQ promotes adipogenesis in an m6A-YTHDF1–dependent manner

BackgroundIntramuscular fat (IMF) content is a critical indicator of pork quality, and abnormal IMF is also relevant to human disease as well as aging. Although N6-methyladenosine (m6A) RNA modification was recently found to regulate adipogenesis in porcine intramuscular fat, however, the underlying molecular mechanisms was still unclear.ResultsIn this work, we collected 20 longissimus dorsi muscle samples with high (average 3.95%) or low IMF content (average 1.22%) from a unique heterogenous swine population for m6A sequencing (m6A-seq). We discovered 70 genes show both differential RNA expression and m6A modification from high and low IMF group, including ADIPOQ and SFRP1, two hub genes inferred through gene co-expression analysis. Particularly, we observed ADIPOQ, which contains three m6A modification sites within 3′ untranslated and protein coding region, could promote porcine intramuscular preadipocyte differentiation in an m6A-dependent manner. Furthermore, we found the YT521‑B homology domain family protein 1 (YTHDF1) could target and promote ADIPOQ mRNA translation.ConclusionsOur study provided a comprehensive profiling of m6A methylation in porcine longissimus dorsi muscle and characterized the involvement of m6A epigenetic modification in the regulation of ADIPOQ mRNA on IMF deposition through an m6A-YTHDF1-dependent manner.

Abstract 6264: NUV-868, a novel BD2-selective BET inhibitor, in combination with enzalutamide or olaparib, inhibits growth of solid tumor xenografts

Abstract The bromodomain and extraterminal domain (BET) family of proteins play a vital role in gene transcription, making it an attractive therapeutic target for cancer. BET inhibitors can also combine with many anticancer agents to enhance activity. The bromodomains of the BET protein, BD1 and BD2, have unique functions and inhibiting either domain can result in differential responses. To date, non-selective BET inhibitors have failed during early clinical development due to significant on-target toxicity and limited benefit; however, selectively targeting specific bromodomains may result in a more favorable benefit/risk profile. NUV-868 is a novel and highly selective BD2 inhibitor of the BET protein family with ~1500-fold selectivity for BRD4-BD2 relative to BRD4-BD1. Herein, we describe NUV-868 and its activity in multiple in vitro and in vivo solid tumor models. Target engagement, selectivity of bromodomain inhibition, and regulation of BET-mediated gene expression were examined. Additionally, the antitumor activity of NUV-868 in combination with enzalutamide or olaparib was studied in tumor xenograft models of prostate, breast, and pancreatic cancer. NUV-868 demonstrated high selectivity for BD2 and regulated expression of several BET target genes. NUV-868 in combination with enzalutamide inhibited growth of several prostate cancer cell line- and patient-derived xenografts. NUV-868 in combination with olaparib inhibited tumor growth in models of breast, ovarian and pancreatic cancer. Our preclinical data demonstrate that NUV-868, a BD2-selective BET inhibitor, inhibits growth of tumor xenografts in combination with enzalutamide or olaparib and provides rationale for examination of these combinations in the clinic. An ongoing, phase 1 clinical study (NCT05252390) is evaluating NUV-868 as a monotherapy and in combination with olaparib or enzalutamide in patients with advanced solid tumors. Citation Format: Hitisha Patel, Jennifer Hertzog, Laura Heller, Spandana Vootukuri, Yan Zhang, Chris Miller, Gary Hattersley. NUV-868, a novel BD2-selective BET inhibitor, in combination with enzalutamide or olaparib, inhibits growth of solid tumor xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6264.

BET Bromodomain Inhibitors: Novel Design Strategies and Therapeutic Applications

The mammalian bromodomain and extra-terminal domain (BET) family of proteins consists of four conserved members (Brd2, Brd3, Brd4, and Brdt) that regulate numerous cancer-related and immunity-associated genes. They are epigenetic readers of histone acetylation with broad specificity. BET proteins are linked to cancer progression due to their interaction with numerous cellular proteins including chromatin-modifying factors, transcription factors, and histone modification enzymes. The spectacular growth in the clinical development of small-molecule BET inhibitors underscores the interest and importance of this protein family as an anticancer target. Current approaches targeting BET proteins for cancer therapy rely on acetylation mimics to block the bromodomains from binding chromatin. However, bromodomain-targeted agents are suffering from dose-limiting toxicities because of their effects on other bromodomain-containing proteins. In this review, we provided an updated summary about the evolution of small-molecule BET inhibitors. The design of bivalent BET inhibitors, kinase and BET dual inhibitors, BET protein proteolysis-targeting chimeras (PROTACs), and Brd4-selective inhibitors are discussed. The novel strategy of targeting the unique C-terminal extra-terminal (ET) domain of BET proteins and its therapeutic significance will also be highlighted. Apart from single agent treatment alone, BET inhibitors have also been combined with other chemotherapeutic modalities for cancer treatment demonstrating favorable clinical outcomes. The investigation of specific biomarkers for predicting the efficacy and resistance of BET inhibitors is needed to fully realize their therapeutic potential in the clinical setting.

Domain shuffling of a highly mutable ligand-binding fold drives adhesin generation across the bacterial kingdom.

Bacterial fibrillar adhesins are specialized extracellular polypeptides that promote the attachment of bacteria to the surfaces of other cells or materials. Adhesin-mediated interactions are critical for the establishment and persistence of stable bacterial populations within diverse environmental niches and are important determinants of virulence. The fibronectin (Fn)-binding fibrillar adhesin CshA, and its paralogue CshB, play important roles in host colonization by the oral commensal and opportunistic pathogen Streptococcus gordonii. As paralogues are often catalysts for functional diversification, we have probed the early stages of structural and functional divergence in Csh proteins by determining the X-ray crystal structure of the CshB adhesive domain NR2 and characterizing its Fn-binding properties in vitro. Despite sharing a common fold, CshB_NR2 displays an ~1.7-fold reduction in Fn-binding affinity relative to CshA_NR2. This correlates with reduced electrostatic charge in the Fn-binding cleft. Complementary bioinformatic studies reveal that homologues of CshA/B_NR2 domains are widely distributed in both Gram-positive and Gram-negative bacteria, where they are found housed within functionally cryptic multi-domain polypeptides. Our findings are consistent with the classification of Csh adhesins and their relatives as members of the recently defined polymer adhesin domain (PAD) family of bacterial proteins.

YTHDF2 Is Downregulated in Response to Host Shutoff Induced by DNA Virus Infection and Regulates Interferon-Stimulated Gene Expression.

Recent studies have begun to reveal the complex and multifunctional roles of N6-methyladenosine (m6A) modifications and their associated writer, reader, and eraser proteins in infection by diverse RNA and DNA viruses. However, little is known about their regulation and functions during infection by several viruses, including poxviruses. Here, we show that members of the YTH Domain Family (YTHDF), in particular YTHDF2, are downregulated as the prototypical poxvirus, vaccinia virus (VacV) enters later stages of replication in a variety of natural target cell types, but not in commonly used transformed cell lines wherein the control of YTHDF2 expression appears to be dysregulated. YTHDF proteins also decreased at late stages of infection by herpes simplex virus 1 (HSV-1) but not human cytomegalovirus, suggesting that YTHDF2 is downregulated in response to infections that induce host shutoff. In line with this idea, YTHDF2 was potently downregulated upon infection with a VacV mutant expressing catalytically inactive forms of the decapping enzymes, D9 and D10, which fails to degrade dsRNA and induces a protein kinase R response that itself inhibits protein synthesis. Overexpression and RNAi-mediated depletion approaches further demonstrate that YTHDF2 does not directly affect VacV replication. Instead, experimental downregulation of YTHDF2 or the related family member, YTHDF1, induces a potent increase in interferon-stimulated gene expression and establishes an antiviral state that suppresses infection by either VacV or HSV-1. Combined, our data suggest that YTHDF2 is destabilized in response to infection-induced host shutoff and serves to augment host antiviral responses. IMPORTANCE There is increasing recognition of the importance of N6-methyladenosine (m6A) modifications to both viral and host mRNAs and the complex roles this modification plays in determining the fate of infection by diverse RNA and DNA viruses. However, in many instances, the functional contributions and importance of specific m6A writer, reader, and eraser proteins remains unknown. Here, we show that natural target cells but not transformed cell lines downregulate the YTH Domain Family (YTHDF) of m6A reader proteins, in particular YTHDF2, in response to shutoff of protein synthesis upon infection with the large DNA viruses, vaccinia virus (VacV), or herpes simplex virus type 1. We further reveal that YTHDF2 downregulation also occurs as part of the host protein kinase R response to a VacV shutoff mutant and that this downregulation of YTHDF family members functions to enhance interferon-stimulated gene expression to create an antiviral state.

CRELD2, endoplasmic reticulum stress, and human diseases.

CRELD2, a member of the cysteine-rich epidermal growth factor-like domain (CRELD) protein family, is both an endoplasmic reticulum (ER)-resident protein and a secretory factor. The expression and secretion of CRELD2 are dramatically induced by ER stress. CRELD2 is ubiquitously expressed in multiple tissues at different levels, suggesting its crucial and diverse roles in different tissues. Recent studies suggest that CRELD2 is associated with cartilage/bone metabolism homeostasis and pathological conditions involving ER stress such as chronic liver diseases, cardiovascular diseases, kidney diseases, and cancer. Herein, we first summarize ER stress and then critically review recent advances in the knowledge of the characteristics and functions of CRELD2 in various human diseases. Furthermore, we highlight challenges and present future directions to elucidate the roles of CRELD2 in human health and disease.

Abstract P4-02-08: Towards Precision Radiation Oncology: Endocrine Therapy Resistance as a Biomarker for Radiation Resistance in ER-Positive Breast Cancer

Abstract Pre-operative endocrine therapy use in post-menopausal women with localized, ER-positive breast cancer affords comparable rates of response and breast preservation, but lower toxicity relative to chemotherapy. Pre-operative endocrine therapy exerts selective pressure on cancer cells and promote evolution and/or enrichment of pathogenic alternations such as ESR1 mutations and other cellular adaptations. How such endocrine therapy-induced adaptations alter response to radiation therapy remains poorly defined. In this study, we show that diverse mechanisms that confer endocrine therapy resistance also drive radiation resistance by reprogramming of DNA repair pathways. We also show that BRD4, a member of bromodomain and extraterminal domain (BET) family of proteins, mediates such DNA repair reprogramming. BRD4 also plays a key role in trascriptional reprogramming in ER-positive breast cancer cells that confers endocrine therapy resistance. Thus, OTX015, a BET inhibitor with a favorable safety profile in early stage clinical trials, reverses both endocrine therapy resistance and radiation resistance in ER-positive breast cancer cells and tumors. Our findings are also consistent with reports that tamoxifen-resistant breast cancer cells are resistant to DNA damaging chemotherapeutic agents such as adriamycin and cisplatin. Overall, our findings suggest that endocrine therapy resistance in the pre-operative setting serves as a biomarker for reduced response to adjuvant radiation therapy, and that pharmacological BET inhibition reverses radiation resistance in such patients. The increasing use of “window of opportunity” studies to assess response to endocrine therapy in the pre-operative setting will further facilitate personalization of radiation treatments in these patients based on their response to endocrine therapy. Thus, we provides a therapeutic rationale for personalization of radiation treatments in breast cancer patients based on their response to endocrine therapy. We also provide a molecularly targeted approach for reversing radiation resistance in such patients. Thus, our study provides a framework for advancing Precision Radiation Oncology in breast cancer patients. Citation Format: SM Nashir Udden, Asal Rahimi, Dong W. Nathan Kim, Prasanna Alluri. Towards Precision Radiation Oncology: Endocrine Therapy Resistance as a Biomarker for Radiation Resistance in ER-Positive Breast Cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-02-08.

HMM-based profiling identifies the binding to divalent cations and nucleotides as common denominators of suramin targets

Introduction: Suramin is one of the pharmacopeia’s most promiscuous drugs. Originally developed for African trypanosomiasis, suramin was also used for onchocerciasis and it has been proposed as an anticancer agent, antiviral drug, therapy for arthritis, autism, and antidote for snake bites. Target proteins of suramin have been described from different species. Here we identify the common motifs among these various targets, aiming to explain the promiscuous nature of suramin.Methods: We have searched for suramin target proteins in the literature and in chemical databases. Applying rigorous inclusion criteria, a list of 44 diverse proteins was assembled with experimental evidence for direct interaction with, and inhibition by, suramin. Hidden Markov model-based target profiling was performed by running the full set of Pfam protein family domains against these proteins.Results: Common denominators were identified by mapping the identified Pfam domains to molecular function gene ontology terms. This in silico pipeline identified nucleotide binding, nucleic acid binding, and binding to divalent cations as the most common denominators of the suramin targets.Discussion: Our results suggest that the extraordinary polypharmacology of suramin may be caused by its ability to inhibit the interaction of proteins with nucleotides or nucleic acids and with divalent cations (Mg2+, Ca2+, Zn2+). Suramin is well known to inhibit nucleotide receptors and nucleic acid-binding enzymes. The association with divalent cations is new and might be key towards the design of better, more selective inhibitors.

A novel high-prevalence antigen in the Lutheran system, LUGA (LU24), and an updated, full-length 3D BCAM model.

The basal cell adhesion molecule (BCAM) carries the antigens of the Lutheran (LU, ISBT005) system. We report a novel Lutheran antigen and propose an updated, full-length 3D model of BCAM. Red blood cell testing, antibody identification, and BCAM genomic DNA sequencing were done by standard methods. Multi-template homology modeling of BCAM used structural templates selected for coverage, highest sequence identity, and protein domain family. All variants causing the loss or gain of a Lutheran antigen were analyzed for residue accessibility and intraprotein interactions. An antibody to a high-prevalence antigen in the plasma of a pregnant woman was determined to be directed at a novel Lutheran antigen. Sequencing of BCAM found three homozygous changes: c.212G > A (p.Arg71His) and two silent, c.711C > T and c.714C > T. The model was built from the first two immunoglobulin crystallized domains of BCAM (D1, D2), three other templates (for D3, D4 and D5 with a higher sequence identity with the target than those used for the model proposed by Burton and Brady in 2008, and for the transmembrane region) and RaptorX (for the intracellular domain). All residues associated with a Lutheran antigen were found to be exposed in wild-type or variant proteins, except p.447 associated with loss of Lu13 expression. The c.212G > A change results in the loss of LUGA (LU24) antigen. Whole genome sequencing continues to reveal polymorphisms with uncertain immunogenicity. This model and demonstration that nearly all residues associated with the expression of a Lutheran antigen are exposed will help evaluate the significance of new polymorphisms.

Elucidating the mechanism of MBD protein LLPS and its role in heterochromatin formation and transcriptional repression.

Membraneless organelles (MLOs) self-assemble into condensed, biochemically distinct microenvironments through liquid-liquid phase separation (LLPS). Heterochromatin, most recently considered an MLO, assembles through weak, multivalent interactions with its associated proteins that contain intrinsically disordered regions. However, the details of the complex molecular interactions between heterochromatin-associated proteins and methylated DNA that drive LLPS have not been fully explored. It is crucial that we elucidate the molecular mechanisms involved in this process as it regulates vital nuclear processes such as transcriptional repression. Its dysregulation is implicated in neurological disorders and cancer. Here, we focus on two members of the methyl-CpG-binding domain (MBD) family of proteins, MBD2 and MBD3, that interpret methylated residues on heterochromatin's underlying DNA. We utilize biochemical and biophysical techniques to (1) determine the conditions and properties that promote MBD2 and MBD3 LLPS and elucidate the molecular mechanism(s) that underpin this process and (2) identify the role binding partners have on MBD2 and MBD3 LLPS. LLPS droplet formation is monitored using UV-Vis spectroscopy and differential interference contrast (DIC) and fluorescence microscopy. To better understand the molecular basis that drives LLPS, small-angle X-ray scattering (SAXS) and dynamic light scattering (DLS) is used to obtain structural and dynamic details of MBD2 and MBD3. Our results provide details into the mechanism(s) by which MBD2 and MBD3 undergo LLPS individually and how this process is enhanced by binding to each other and methylated DNA. Uncovering the driving forces that assemble MBD protein-based LLPS droplets will give us insight into the higher-order organization of heterochromatin and how it functions within this structure. Additionally, understanding how disease-related mutations lead to aberrant formation of condensates or the inability to form condensates will provide novel therapeutic targets.

Selected Articles from This Issue

Highlights| February 01 2023 Selected Articles from This Issue Author & Article Information Online Issn: 1557-3125 Print Issn: 1541-7786 ©2023 American Association for Cancer Research2023American Association for Cancer Research Mol Cancer Res (2023) 21 (2): 89. https://doi.org/10.1158/1541-7786.MCR-21-2-HI Related Content A commentary has been published: BET Inhibitors Target the SCLC-N Subtype of Small-Cell Lung Cancer by Blocking NEUROD1 Transactivation A commentary has been published: Downregulation of iNOS/NO Promotes Epithelial–Mesenchymal Transition and Metastasis in Colorectal Cancer A commentary has been published: TWEAK–Fn14–RelB Signaling Cascade Promotes Stem Cell–like Features that Contribute to Post-Chemotherapy Ovarian Cancer Relapse View more A commentary has been published: XIAP and PHB1 Regulate Anoikis through Competitive Binding to TRAF6 View less Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Tools Icon Tools Get Permissions Cite Icon Cite Search Site Article Versions Icon Versions Version of Record February 1 2023 Citation Selected Articles from This Issue. Mol Cancer Res 1 February 2023; 21 (2): 89. https://doi.org/10.1158/1541-7786.MCR-21-2-HI Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest Search Advanced Search NEUROD1 is the master transcriptional regulator defining the small-cell lung cancer (SCLC) SCLC-N subtype, but NEUROD1 has proven difficult to therapeutically target. Targeting NEUROD1 transcriptional coactivators may be an effective therapeutic approach, but such coactivators have yet to be defined. To identify NEUROD1 transcriptional coactivators, Chen and colleagues elucidated NEUROD1 genomic localization regions using ChIP-Seq and searched for colocalizing coactivator candidates using a ChIP-Seq database. The authors discovered that BRD4, a member of the bromodomain and extraterminal domain (BET) protein family, significantly colocalizes to NEUROD1-inhabited genomic regions. Co-immunoprecipitation experiments demonstrated that BRD4 and NEUROD1 interact, and treatment with BET inhibitors (BETi) decreases NEUROD1 target gene expression, suggesting BRD4 is a NEUROD1 transcriptional coactivator. Accordingly, the authors found that SCLC-N cells are more sensitive to BETi than are other SCLC subtype cells, and that BETi significantly slow SCLC-N patient-derived xenograft tumor growth in mouse models. Using a siRNA screen, the authors... You do not currently have access to this content.