Specific Binding Proteins Research Articles

Selective Hydroxylation of C(sp3 )-H Bonds in Steroids.

Steroids are highly prevalent structures in small-molecule therapeutics, with the level of oxidation being key to their biological activity and physicochemical properties. These C(sp3 )-rich tetracycles contain many stereocentres, which are important for creating specific vectors and protein binding orientations. Therefore, the ability to hydroxylate steroids with a high degree of regio-, chemo- and stereoselectivity is essential for researchers working in this field. This review will cover three main methods for the hydroxylation of steroidal C(sp3 )-H bonds: biocatalysis, metal-catalysed C-H hydroxylation and organic oxidants, such as dioxiranes and oxaziridines.

Mapping Peptide-Protein Interactions by Amine-Reactive Cleavable Photoaffinity Reagents.

Photoaffinity labeling followed by tandem mass spectrometry is an often used strategy to identify protein targets of small-molecule drugs or drug candidates, which, under ideal conditions, enables the identification of the actual drug binding site. In the case of bioactive peptides, however, identifying the distinct binding site is hampered because of complex fragmentation patterns during tandem mass spectrometry. We here report the development and use of small cleavable photoaffinity reagents that allow functionalization of bioactive peptides for light-induced covalent binding to their protein targets. Upon cleavage of the covalently linked peptide drug, a chemical remnant of a defined mass remains on the bound amino acid, which is then used to unambiguously identify the drug binding site. Applying our approach to known peptide-drug/protein pairs with reported crystal structures, such as the calmodulin-melittin interaction, we were able to validate the identified binding sites based on structural models. Overall, our cleavable photoaffinity labeling strategy represents a powerful tool to enable the identification of protein targets and specific binding sites of a wide variety of bioactive peptides in the future.

Impact of Manganese and Chromate on Specific DNA Double-Strand Break Repair Pathways.

Manganese is an essential trace element; nevertheless, on conditions of overload, it becomes toxic, with neurotoxicity being the main concern. Chromate is a well-known human carcinogen. The underlying mechanisms seem to be oxidative stress as well as direct DNA damage in the case of chromate, but also interactions with DNA repair systems in both cases. However, the impact of manganese and chromate on DNA double-strand break (DSB) repair pathways is largely unknown. In the present study, we examined the induction of DSB as well as the effect on specific DNA DSB repair mechanisms, namely homologous recombination (HR), non-homologous end joining (NHEJ), single strand annealing (SSA), and microhomology-mediated end joining (MMEJ). We applied DSB repair pathway-specific reporter cell lines, pulsed field gel electrophoresis as well as gene expression analysis, and investigated the binding of specific DNA repair proteins via immunoflourescence. While manganese did not seem to induce DNA DSB and had no impact on NHEJ and MMEJ, HR and SSA were inhibited. In the case of chromate, the induction of DSB was further supported. Regarding DSB repair, no inhibition was seen in the case of NHEJ and SSA, but HR was diminished and MMEJ was activated in a pronounced manner. The results indicate a specific inhibition of error-free HR by manganese and chromate, with a shift towards error-prone DSB repair mechanisms in both cases. These observations suggest the induction of genomic instability and may explain the microsatellite instability involved in chromate-induced carcinogenicity.

PZR promotes tumorigenicity of lung cancer cells by regulating cell migration and invasion via modulating oxidative stress and cell adhesion.

PZR is a transmembrane glycoprotein encoded by the MPZL1 gene. It serves as a specific binding protein and substrate of tyrosine phosphatase SHP-2 whose mutations cause developmental diseases and cancers. Bioinformatic analyses of cancer gene databases revealed that PZR is overexpressed in lung cancer and correlated with unfavorable prognosis. To investigate the role of PZR in lung cancer, we employed the CRISPR technique to knockout its expression and recombinant lentiviruses to overexpress it in lung adenocarcinoma SPC-A1 cells. While knockout of PZR reduced colony formation, migration, and invasion, overexpression of PZR had the opposite effects. Furthermore, when implanted in immunodeficient mice, PZR-knockout SPC-A1 cells showed suppressed tumor-forming ability. Finally, the underlying molecular mechanism for these functions of PZR is its positive role in activating tyrosine kinases FAK and c-Src and in maintaining the intracellular level of reactive oxygen species (ROS). In conclusion, our data indicated that PZR plays an important role in lung cancer development, and it may serve as a therapeutic target for anti-cancer development and as a biomarker for cancer prognosis.

Finding motifs using DNA images derived from sparse representations.

Motifs play a crucial role in computational biology, as they provide valuable information about the binding specificity of proteins. However, conventional motif discovery methods typically rely on simple combinatoric or probabilistic approaches, which can be biased by heuristics such as substring-masking for multiple motif discovery. In recent years, deep neural networks have become increasingly popular for motif discovery, as they are capable of capturing complex patterns in data. Nonetheless, inferring motifs from neural networks remains a challenging problem, both from a modeling and computational standpoint, despite the success of these networks in supervised learning tasks. We present a principled representation learning approach based on a hierarchical sparse representation for motif discovery. Our method effectively discovers gapped, long, or overlapping motifs that we show to commonly exist in next-generation sequencing datasets, in addition to the short and enriched primary binding sites. Our model is fully interpretable, fast, and capable of capturing motifs in a large number of DNA strings. A key concept emerged from our approach-enumerating at the image level-effectively overcomes the k-mers paradigm, enabling modest computational resources for capturing the long and varied but conserved patterns, in addition to capturing the primary binding sites. Our method is available as a Julia package under the MIT license at https://github.com/kchu25/MOTIFs.jl, and the results on experimental data can be found at https://zenodo.org/record/7783033.

Extracellular vesicle-microRNAs mediated response of bovine ovaries to seasonal environmental changes

BackgroundAmong the various seasonal environmental changes, elevated ambient temperature during the summer season is a main cause of stress in dairy and beef cows, leading to impaired reproductive function and fertility. Follicular fluid extracellular vesicles (FF-EVs) play an important role in intrafollicular cellular communication by, in part, mediating the deleterious effects of heat stress (HS). Here we aimed to investigate the changes in FF-EV miRNA cargoes in beef cows in response to seasonal changes: summer (SUM) compared to the winter (WIN) season using high throughput sequencing of FF-EV-coupled miRNAs. In addition to their biological relevance, the potential mechanisms involved in the packaging and release of those miRNAs as a response to environmental HS were elucidated.ResultsSequencing analysis revealed that an average of 6.6% of the EV-RNA mapped reads were annotated to bovine miRNAs. Interestingly, miR-148a, miR-99a-5p, miR-10b, and miR-143 were the top four miRNAs in both groups accounting for approximately 52 and 62% of the total miRNA sequence reads in the SUM and WIN groups, respectively. A group of 16 miRNAs was up-regulated and 8 miRNAs were down-regulated in the SUM compared to the WIN group. Five DE-miRNAs (miR-10a, miR-10b, miR-26a, let-7f, and miR-1246) were among the top 20 expressed miRNA lists. Sequence motif analysis revealed the appearance of two specific motifs in 13 out of the 16 upregulated miRNAs under HS conditions. Both motifs were found to be potentially bonded by specific RNA binding proteins including Y-box binding proteins (YBX1 and YBX2) and RBM42.ConclusionOur findings indicate that FF EV-coupled miRNA profile varies under seasonal changes. These miRNAs could be a good indicator of the cellular mechanism in mediating HS response and the potential interplay between miRNA motifs and RNA binding proteins can be one of the mechanisms governing the packaging and release of miRNAs via EVs to facilitate cellular survival.

Abstract A031: KRAS (G12C) mediated mRNA translation program

Abstract KRAS-dependent activation of mRNA translation signaling is a common feature in cancer however, the KRAS-specific translation landscape and functional proteome are not fully characterized in a genome-wide manner. We show that mutant KRAS drives specific mechanisms of translational control to support cancer growth. We have utilized the technology of ribosome footprinting combined with advanced sequencing methodology to define the translation landscape affected by a specific inhibitor of KRAS, sotorasib that targets G12C mutant KRAS activity. We show that acute treatment of sotorasib inhibits the translation of key oncogenic proteins including MYC and MYC targets. KRAS (G12C) inhibition profoundly affects the translation of ribosomal proteins and translation elongation factors. G12C mutant KRAS remarkably affects the translation of proteins involved in the ROS and DNA repair pathways in cancer. Moreover, these translational changes are mediated through specific RNA motifs and RNA binding proteins. In summary, our study defines the KRAS (G12C) specific regulation of translation and provides the mechanism of KRAS (G12C) mediated regulation of cancer proteome. Citation Format: Kamini Singh. KRAS (G12C) mediated mRNA translation program [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A031.

Abstract B036: Treatment modalities in KRAS-driven cancer

Abstract KRAS is the most common RAS isoform, found to be altered in 22% of RAS-mutated cancers. The mutant protein is found most prevalently in pancreatic cancer followed by colorectal, non-small cell lung cancer and other solid tumors. Due to its structural complexity, KRAS remained “undruggable” for several decades. Recently, the discovery of a specific protein binding pocket has led to the development of small molecule inhibitors that potently bind and inhibit KRAS downstream signalling. The accelerated FDA approval of sotorasib and adagrasib for treatment of NSCLC patients harbouring KRASG12C-mutation and further clinical evaluation of several other inhibitors of KRASG12C are under investigation, however emergence of resistance and limited efficacy beyond non-small cell lung cancer is a major concern. Targeting KRAS mutations other than KRASG12C (i.e., without a mutant-specific cysteine residue) with small molecules, has been considered much more challenging, especially for developing covalent inhibitors. Small molecule targeting of KRASG12D (e.g. MRTX1133), is under active preclinical investigation and small molecule drugs that target other KRAS-activating mutations (G12S, G12R and G12V) are being intensely explored. A variety of novel alternative approaches are also being investigated to target tumors driven by mutant KRAS, such as (i) targeting complementary signalling pathways (ii) enhancing mutant KRAS protein degradation using PROTACS, (iii) developing pan-KRAS small molecules inhibitors, (iv) anti-KRAS immune cell-based therapies, (v) small interfering RNAs to block mutant KRAS expression and (vi) anti-KRAS vaccines. Here, we aim to provide a comprehensive overview of current status and future prospect of these modalities, in the context of RAS-mutated cancers. Citation Format: Darren R. Tyson, Neetu Singh, Prashant K. Bhavar, Partha Pratim Sarma, Uday Kumar Surampudi, Sathish Katike. Treatment modalities in KRAS-driven cancer [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B036.

Liquid crystal-assisted optical biosensor for early-stage diagnosis of mammary glands using HER-2

Breast cancer (BC) is one of the most commonly diagnosed cancers and the second leading cause of cancer mortality among women around the world. The purpose of this study is to present a non-labeled liquid crystal (LC) biosensor, based on the inherent feature of nematic LCs, for the evaluation of BC using the human epidermal growth factor receptor-2 (HER-2) biomarker. The mechanism of this sensing is supported by surface modification with dimethyloctadecyl [3-(trimethoxysilyl) propyl] ammonium chloride (DMOAP) encouraging the long alkyl chains that induce a homeotropic orientation of the LC molecules at the interface. To enhance the binding efficacy of more HER-2 antibody (Ab) on LC aligning agents, a simple ultraviolet radiation-assisted method was also used to increase functional groups on the DMOAP coated slides, thereby improving binding affinity and efficiency onto HER-2 Abs. The designed biosensor makes use of the specific binding of HER-2 protein to HER-2 Ab and disruption of the orientation of LCs. This orientation change leads to a transition of the optical appearance from dark to birefringent, enabling the detection of HER-2. This novel biosensor exhibits a linear optical response to HER-2 concentration in the wide dynamic range of 10–6–102 ng/mL, with an ultra-low detection limit of 1 fg/mL. As a proof of concept, the designed LC biosensor was successfully investigated for the quantification of HER-2 protein in patients suffering from BC. Owing to the sensitivity, selectivity, and label-free detection, this biosensor may amplify the application of LC-based biosensors for the detection of most types of cancers.

Abstract 6205: Visualizing dynamics of long non-coding RNA TUG1 in live glioblastoma cells

Abstract Long non-coding RNAs (lncRNAs) are transcripts longer than 200 nucleotides that are not translated into proteins. lncRNA TUG1 (taurine upregulated gene 1) is highly expressed in various cancers, including glioblastoma. Previously we found that TUG1 is an essential molecule for cancer cells to cope with excessive replication stress in the S phase. Albeit its importance to cell cycle-related function, how it is dynamically regulated during the cell cycle is entirely unknown. Here, we established a system visualizing endogenous TUG1 molecules in live cells. TUG1 was tagged with stem-loop motifs recognized by a specific binding protein fused with GFP. We tracked TUG1 RNA by the GFP signal for 22 hours in T98G glioblastoma cells and found that the expression started from the later G1 phase. When cells were treated with Hydroxy Urea, a chemical that interferes with replication and increases replication stress, the expression of TUG1 was further upregulated in the S phase within 20 mins. We also observed some TUG1 molecules accumulated in several nuclear locations. It may indicate that TUG1 assembles to protect damaged DNA loci efficiently. Our results suggest that the expression of TUG1 is regulated in a cell cycle-dependent manner and is strongly connected with its function of TUG1. The current live-cell imaging system could help further discover the critical roles of TUG1 in cancer cells. Citation Format: Ayaka Minematsu. Visualizing dynamics of long non-coding RNA TUG1 in live glioblastoma cells [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 6205.

Insulin-like growth factor binding Protein-4: A novel indicator of pulmonary arterial hypertension severity and survival.

Proteomic analysis of patients with pulmonary arterial hypertension (PAH) has demonstrated significant abnormalities in the insulin-like growth factor axis (IGF). This study proposed to establish associations between a specific binding protein, insulin-like growth factor binding protein 4 (IGFBP4), and PAH severity as well as survival across varying study cohorts. In all cohorts studied, serum IGFBP4 levels were significantly elevated in PAH compared to controls (p < 0.0001). IGFBP4 concentration was also highest in the connective tissue-associated PAH (CTD-PAH) and idiopathic PAH subtypes (876 and 784 ng/mL, median, respectively). After adjustment for age and sex, IGFBP4 was significantly associated with worse PAH severity as defined by a decreased 6-min walk distance (6MWD), New York heart association functional class (NYHA-FC), REVEAL 2.0 score and higher right atrial pressures. In longitudinal analysis provided by one of the study cohorts, IGFBP4 was prospectively significantly associated with a shorter 6MWD, worse NYHA-FC classification, and decreased survival. Cox multivariable analysis demonstrated higher serum IGFBP4 as an independent predictor of survival in the overall PAHB cohort. Therefore, this study established that higher circulating IGFBP4 levels were significantly associated with worse PAH severity, decreased survival and disease progression. Dysregulation of IGF metabolism/growth axis may play a significant role in PAH cardio-pulmonary pathobiology.

Investigation of the mechanism of action of mefloquine and derivatives against the parasite Echinococcus multilocularis

Alveolar echinococcosis (AE) is caused by infection with the fox tapeworm E. multilocularis. The disease affects humans, dogs, captive monkeys, and other mammals, and it is caused by the metacestode stage of the parasite growing invasively in the liver. The current drug treatment is based on non-parasiticidal benzimidazoles. Thus, they are only limitedly curative and can cause severe side effects. Therefore, novel and improved treatment options for AE are needed.Mefloquine (MEF), an antimalarial agent, was previously shown to be effective against E. multilocularis in vitro and in experimentally infected mice. However, MEF is not parasiticidal and needs improvement for successful treatment of patients, and it can induce strong neuropsychiatric side-effects. In this study, the structure-activity relationship and mode of action of MEF was investigated by comparative analysis of 14 MEF derivatives. None of them showed higher activity against E. multilocularis metacestodes compared to MEF, but four compounds caused limited damage. In order to identify molecular targets of MEF and effective derivatives, differential affinity chromatography combined with mass spectrometry was performed with two effective compounds (MEF, MEF-3) and two ineffective compounds (MEF-13, MEF-22). 1′681 proteins were identified that bound specifically to MEF or derivatives. 216 proteins were identified as binding only to MEF and MEF-3. GO term enrichment analysis of these proteins and functional grouping of the 25 most abundant MEF and MEF-3 specific binding proteins revealed the key processes energy metabolism and cellular transport and structure, as well as stress responses and nucleic acid binding to be involved. The previously described ferritin was confirmed as an exclusively MEF-binding protein that could be relevant for its efficacy against E. multilocularis. The here identified potential targets of MEF will be further investigated in the future for a clear understanding of the pleiotropic effects of MEF, and improved therapeutic options against AE.

Pseudorabies virus exploits N6-methyladenosine modification to promote viral replication.

Pseudorabies virus (PRV) is the pathogenic virus of porcine pseudorabies (PR), belonging to the Herpesviridae family. PRV has a wide range of hosts and in recent years has also been reported to infect humans. N6-methyladenosine (m6A) modification is the major pathway of RNA post-transcriptional modification. Whether m6A modification participates in the regulation of PRV replication is unknown. Here, we investigated that the m6A modification was abundant in the PRV transcripts and PRV infection affected the epitranscriptome of host cells. Knockdown of cellular m6A methyltransferases METTL3 and METTL14 and the specific binding proteins YTHDF2 and YTHDF3 inhibited PRV replication, while silencing of demethylase ALKBH5 promoted PRV output. The overexpression of METTL14 induced more efficient virus proliferation in PRV-infected PK15 cells. Inhibition of m6A modification by 3-deazaadenosine (3-DAA), a m6A modification inhibitor, could significantly reduce viral replication. Taken together, m6A modification played a positive role in the regulation of PRV replication and gene expression. Our research revealed m6A modification sites in PRV transcripts and determined that m6A modification dynamically mediated the interaction between PRV and host.

Decreased IgE Binding and Less Allergenic Protein Per Unit Mass in Bamba Peanut Snack Compared to Crushed Peanut

Substrates for peanut oral immunotherapy (OIT) vary, including crushed peanut and Bamba peanut snack (Osem, Israel). While the total peanut protein content in each food has been estimated, specific IgE binding levels, protein extractability, and relative allergen content have not been compared.

A Lambda-Exonuclease SELEX Method for Generating Aptamers to Bacterial Targets.

Nucleic acid aptamers are short sequences of single-stranded (ss) DNA or RNA that fold into a three-dimensional shape with useful binding properties. Traditionally, these properties have included specific recognition and binding of ions, small-molecules, proteins, and enzyme targets. Increasingly though, aptamers are being raised against complex subcellular or cellular targets. These broader-affinity aptamers can be usefully employed for detection, labeling, or therapeutic targeting of intact/living cells, whether prokaryotic or eukaryotic. Aptamers are usually developed from a random-sequence oligonucleotide library by repeated rounds of selection and amplification, a process named "systematic evolution of ligands by exponential enrichment" (SELEX). We describe here a widely applicable cell-SELEX method for raising aptamers against bacteria, using Escherichia coli strain HB101 as an example. Our cell-SELEX method uses a cycle of four stages: (1) incubation of a fluorescently labeled random-sequence ssDNA library with bacterial cells; (2) separation of cell-associated ssDNA from free ssDNA; (3) amplification of bound ssDNA by PCR, and (4) use of lambda-exonuclease to selectively regenerate ssDNA for further rounds of selection.

PS-BPB05-8: POSSIBLE INTERACTION OF ANGIOTENSIN II TYPE 1 RECEPTOR-ASSOCIATED PROTEIN WITH TRANSFERRIN RECEPTOR-1 VIA MODULATION OF THE RECEPTOR INTERNALIZATION

Objective: Angiotensin II type 1 receptor (AT1R)-associated protein (ATRAP) was originally identified as a specific binding protein of AT1R. We have shown that ATRAP promotes constitutive internalization of AT1R so as to inhibit the pathological activation of its downstream signaling. Also, we reported that genetic knockdown of ATRAP exacerbates kidney fibrosis in mice along with functional mitochondrial abnormalities and subsequent increases in ROS production. These effects of ATRAP on kidney fibrosis were suggested to be AT1R-independent actions, but the molecular mechanism is unclear. The present study was designed to explore a novel interacting protein of ATRAP that was involved in the mechanism by which ATRAP protected against kidney fibrosis, independent of the interaction with AT1R. Design and method: We established Human Embryonic Kidney 293 cells which were able to induce the expression of Flag-tagged ATRAP (HEK293_Flag-ATRAP cells) in a doxycycline-dependent manner. After immunoprecipitation with anti-Flag antibodies, the Flag-ATRAP complex was analyzed with a mass spectrometer. Among identified proteins, we focused on transferrin receptor1(TfR1). To confirm the molecular interaction, co-immunoprecipitation was performed using anti-Flag or anti-TfR1 antibodies. Additionally, to validate functional interactions, we analyzed intracellular iron concentrations using fluorescent probe of ferric iron. Furthermore, we verified the TfR1 expression in two ways. First, immunofluorescence staining of TfR1 in the whole cell or on the cell surface only. Second, Western blot with whole cell lysate treated with ferristatin II which degraded TfR1 on the cell surface. Results: On the mass spectrometric analysis, 377 proteins were identified as the ATRAP binding protein candidates. Enrichment analysis indicated that proteins related to endocytosis and vesicle trafficking were enriched. TfR1 is also identified. We confirmed the molecular interaction between ATRAP and TfR1 by co-immunoprecipitation. Enhanced ATRAP expression decreased the cellular iron level as well as the expression of TfR1 on the cell surface, despite no evident change in whole cell. Furthermore, we showed that ATRAP suppressed the effect of ferristatin II. Conclusion: We propose a molecular and functional link between ATRAP and TfR1. ATRAP would regulate TfR1 availability via downregulation of cell surface TfR1 via promotion of its internalization. TfR1 promotes intracellular localization of the ferric iron-bound transferrin. Iron is a key factor in the process of kidney fibrosis via production of ROS in relation to deterioration of mitochondrial function. Taken together, this novel ATRAP-TfR1 axis might be the mechanism relevant to the ROS/mitochondrial dysfunction-mediated process of kidney fibrosis.

2129. Maternal Transmission of SARS-CoV-2-specific Antibodies, but not Cytokines/Chemokines to Neonates Following Infection and Vaccination During Pregnancy

Abstract Background Despite extensive studies of human immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and coronavirus disease 2019 (COVID-19) vaccination, research examining protective correlates of vertical transmission following maternal exposure in pregnancy remain limited. Here, we characterized antibody and cytokine responses in maternal and cord blood following infection or vaccination at various timepoints during gestation. Methods Spike S1 protein-specific binding antibodies and antibodies capable of blocking the interaction between the receptor binding domain (RBD) and the angiotensin converting enzyme 2 (ACE2) were measured in maternal and cord blood by ELISA. Serum concentrations of 74 cytokines/chemokines were measured by multiplex assay. Humoral responses and cytokine levels from matched maternal and fetal cord sera were compared and examined for potential correlations. Results We observed a highly significant correlation between Spike S1-specific antibody titer and RBD-ACE2 blocking antibody activity between maternal and fetal cord serum (p &amp;lt; 2.2e-16, R &amp;gt; 0.90). Blocking antibody activity was significantly higher for mothers infected during the 3rd trimester compared to earlier trimesters; however, vaccinated mothers developed and transferred higher antibody titers with greater RBD-ACE2 blocking antibody activity to their neonates than infected mothers. Furthermore, vaccine-induced Spike S1 IgG transfer ratios (fetal cord/maternal) were significantly higher than those induced by infection (p = 0.002). Multiplex assay showed significantly elevated levels of 33 cytokines/chemokines, mainly pro-inflammatory in infected maternal serum samples, while the paired fetal cord samples exhibited an anti-inflammatory cytokine predominance. Conclusion Our data support selective vertical transmission of potentially protective humoral responses against SARS-CoV-2, especially following vaccination in the 3rd trimester. The anti-inflammatory cytokine predominance in cord blood that persists despite maternal SARS-CoV-2 infection may offset the adverse outcomes of inflammation in pregnancy for the neonate. Disclosures All Authors: No reported disclosures.

'Shared-Hook' and 'Changed-Hook' Binding Activities of Herpesviral Core Nuclear Egress Complexes Identified by Random Mutagenesis.

Herpesviruses replicate their genomes and assemble their capsids in the host cell nucleus. To progress towards morphogenesis in the cytoplasm, herpesviruses evolved the strategy of nuclear egress as a highly regulated process of nucleo-cytoplasmic capsid transition. The process is conserved among α-, β- and γ-herpesviruses and involves the formation of a core and multicomponent nuclear egress complex (NEC). Core NEC is assembled by the interaction between the nucleoplasmic hook protein, i.e., pUL53 (human cytomegalovirus, HCMV), and the integral membrane-associated groove protein, i.e., pUL50. Our study aimed at the question of whether a panherpesviral NEC scaffold may enable hook-into-groove interaction across herpesviral subfamilies. For this purpose, NEC constructs were generated for members of all three subfamilies and analyzed for multi-ligand interaction using a yeast two-hybrid (Y2H) approach with randomized pUL53 mutagenesis libraries. The screening identified ten library clones displaying cross-viral shared hook-into-groove interaction. Interestingly, a slightly modified Y2H screening strategy provided thirteen further changed-hook pUL53 clones having lost parental pUL50 interaction but gained homolog interaction. In addition, we designed a sequence-predicted hybrid construct based on HCMV and Epstein-Barr virus (EBV) core NEC proteins and identified a cross-viral interaction phenotype. Confirmation was provided by applying protein-protein interaction analyses in human cells, such as coimmunoprecipitation settings, confocal nuclear rim colocalization assays, and HCMV ΔUL53 infection experiments with pUL53-complementing cells. Combined, the study provided the first examples of cross-viral NEC interaction patterns and revealed a higher yield of human cell-confirmed binding clones using a library exchange rate of 3.4 than 2.7. Thus, the study provides improved insights into herpesviral NEC protein binding specificities of core NEC formation. This novel information might be exploited to gain a potential target scaffold for the development of broadly acting NEC-directed inhibitory small molecules.

DDB2 represses epithelial-to-mesenchymal transition and sensitizes pancreatic ductal adenocarcinoma cells to chemotherapy.

Damage specific DNA binding protein 2 (DDB2) is an UV-indiced DNA damage recognition factor and regulator of cancer development and progression. DDB2 has dual roles in several cancers, either as an oncogene or as a tumor suppressor gene, depending on cancer localization. Here, we investigated the unresolved role of DDB2 in pancreatic ductal adenocarcinoma (PDAC). The expression level of DDB2 in pancreatic cancer tissues and its correlation with patient survival were evaluated using publicly available data. Two PDAC cell models with CRISPR-modified DDB2 expression were developed: DDB2 was repressed in DDB2-high T3M4 cells (T3M4 DDB2-low) while DDB2 was overexpressed in DDB2-low Capan-2 cells (Capan-2 DDB2-high). Immunofluorescence and qPCR assays were used to investigate epithelial-to-mesenchymal transition (EMT) in these models. Migration and invasion properties of the cells were also determined using wound healing and transwell assays. Sensitivity to 5-fluorouracil (5-FU), oxaliplatin, irinotecan and gemcitabine were finally investigated by crystal violet assays. DDB2 expression level was reduced in PDAC tissues compared to normal ones and DDB2-low levels were correlated to shorter disease-free survival in PDAC patients. DDB2 overexpression increased expression of E-cadherin epithelial marker, and decreased levels of N-cadherin mesenchymal marker. Conversely, we observed opposite effects in DDB2 repression and enhanced transcription of SNAIL, ZEB1, and TWIST EMT transcription factors (EMT-TFs). Study of migration and invasion revealed that these properties were negatively correlated with DDB2 expression in both cell models. DDB2 overexpression sensitized cells to 5-fluorouracil, oxaliplatin and gemcitabine. Our study highlights the potential tumor suppressive effects of DDB2 on PDAC progression. DDB2 could thus represent a promising therapeutic target or biomarker for defining prognosis and predicting chemotherapy response in patients with PDAC.

The miRNA125a-5p and miRNA125b-1-5p cluster induces cell invasion by down-regulating DDB2-reduced epithelial-to-mesenchymal transition (EMT) in colorectal cancer

MicroRNA (miRNA) is a kind of non-coding RNA that regulates gene expression and is involved in tumor development. MiRNA-125 is reportedly aberrantly expressed in colorectal cancer tissue; however, its potential function and underlying mechanism remain unclear. The present study aimed to investigate the expression level and potential role of the miRNA-125 family in the invasion and migration of colorectal cancer. To further understand the role of the miRNA-125 family in metastatic colorectal cancer, we overexpressed miRNA-125 in the SW480 cell line by transfection with the miRNA-125 family mimics or a sponge. Methyl thiazolyl tetrazolium (MTT) assay was performed to identify the effect of the miRNA-125 family on cell proliferation, and a Transwell filter assay was used to detect the role of the miRNA-125 family in migration and invasion. A luciferase assay was carried out to confirm the binding site of miRNA-125 and the target gene, damage specific DNA binding protein 2 (DDB2). Western blot was applied to detect the expression levels of DDB2 and the markers of epithelial-to-mesenchymal transition (EMT) in colorectal cancer cells. The real-time polymerase chain reaction (PCR) results showed that miR-125a-5p and miR-125b-1-5p were up-regulated in metastatic colorectal cancer tissues. The Transwell filter assay results appeared that miR-125a-5p and miR-125b-1-5p could promote the invasion and migration of colorectal cancer cells. The luciferase assay data confirmed the binding site of miR-125a-5p and miR-125b-1-5p on the 3' untranslated region (3'UTR) of DDB2 messenger RNA (mRNA). The real-time PCR and Western blot results indicated that miR-125a-5p and miR-125b-1-5p could regulate the expression levels of DDB2 and EMT markers, and lower DDB2 expression was observed in metastatic tissues. Our findings illustrated that miRNA125a-5p and miRNA125b-1-5p could reduce the expression of DDB2 by binding to the 3'UTR region, and then regulate the expression levels of EMT markers, leading to the enhanced invasion and metastasis of colorectal cancer cells. Thus, miRNA125a-5p and miRNA125b-1-5p might be novel markers of colorectal cancer migration and potential therapeutic targets to treat metastatic colorectal cancer patients.