Genome-wide characterization and expression analysis of the HD-Zip II gene family in response to drought and GA3 stresses in Nicotiana tabacum.

BACKGROUND: Homeodomain-leucine ZIPper (HD-ZIP) transcription factors play crucial roles in plant growth, development, and stress responses. The HD-ZIP family is categorised into four groups (HD-ZIP I-IV). While extensive genome-wide studies have been conducted on the HD-ZIP I, III, and IV subfamily in Nicotiana tabacum (tobacco), comprehensive reports on the HD-ZIP II subfamily genes are limited. METHODS: Bioinformatics resources and tools were utilised to analyse molecular characteristics, phylogenetic homology, and protein interactions. Expression pattern analyses in various tissues and the relative expression of NtHD-ZIP II genes under drought and GA3 treatment were assessed by qRT-PCR. RESULTS: In this study, 24 HD-ZIP II members were systematically identified and categorised into seven independent clades through phylogenetic analysis involving tobacco and other plant species. We found that 19 NtHD-ZIP II genes exhibited tissue-specific expression. The transcripts of NtHD-ZIPII3, 4, 14, 23, 24 were notably induced under the drought treatments, while those of NtHD-ZIPII7, 11, 12, 20 were suppressed. Furthermore, NtHD-ZIPII15 transcripts decreased following GA3 treatment, whereas the transcripts of NtHD-ZIPII7, 8, 11, 12 were induced after GA3 treatment. Notably, an increase in trichomes was observed in tobacco leaves treated with GA3 and subjected to drought. CONCLUSIONS: The expression levels of some HD-ZIP II genes were altered, and an increase in glandular trichomes was induced under GA3 and drought treatments in tobacco. Overall, our findings provide insights into the expression patterns of NtHD-ZIP II genes and will facilitate their functional characterisation in future studies.

HD-Zip proteins modify floral structures for self-pollination in tomato.

Cleistogamy is a type of self-pollination that relies on the formation of a stigma-enclosing floral structure. We identify three homeodomain-leucine zipper IV (HD-Zip IV) genes that coordinately promote the formation of interlocking trichomes at the anther margin to unite neighboring anthers, generating a closed anther cone and cleistogamy (flower morphology necessitating strict self-pollination). These HD-Zip IV genes also control style length by regulating the transition from cell division to endoreduplication. The expression of these HD-Zip IV genes and their downstream gene, Style 2.1, was sequentially modified to shape the cleistogamy morphology during tomato evolution and domestication. Our results provide insights into the molecular basis of cleistogamy in modern tomato and suggest targets for improving fruit set and preventing pollen contamination in genetically modified crops.

The Association between the rs3747406 Polymorphism in the Glucocorticoid-Induced Leucine Zipper Gene and Sepsis Survivals Depends on the SOFA Score.

The variability in mortality in sepsis could be a consequence of genetic variability. The glucocorticoid system and the intermediate TSC22D3 gene product-glucocorticoid-induced leucine zipper-are clinically relevant in sepsis, which is why this study aimed to clarify whether TSC22D3 gene polymorphisms contribute to the variance in sepsis mortality. Blood samples for DNA extraction were obtained from 455 patients with a sepsis diagnosis according to the Sepsis-III criteria and from 73 control subjects. A SNP TaqMan assay was used to detect single-nucleotide polymorphisms (SNPs) in the TSC22D3 gene. Statistical and graphical analyses were performed using the SPSS Statistics and GraphPad Prism software. C-allele carriers of rs3747406 have a 2.07-fold higher mortality rate when the sequential organ failure assessment (SOFA) score is higher than eight. In a multivariate COX regression model, the SNP rs3747406 with a SOFA score ≥ 8 was found to be an independent risk factor for 30-day survival in sepsis. The HR was calculated to be 2.12, with a p-value of 0.011. The wild-type allele was present in four out of six SNPs in our cohort. The promoter of TSC22D3 was found to be highly conserved. However, we discovered that the C-allele of rs3747406 poses a risk for sepsis mortality for SOFA Scores higher than 6.

The response of Dual-leucine zipper kinase (DLK) to nocodazole: Evidence for a homeostatic cytoskeletal repair mechanism.

Genetic and pharmacological perturbation of the cytoskeleton enhances the regenerative potential of neurons. This response requires Dual-leucine Zipper Kinase (DLK), a neuronal stress sensor that is a central regulator of axon regeneration and degeneration. The damage and repair aspects of this response are reminiscent of other cellular homeostatic systems, suggesting that a cytoskeletal homeostatic response exists. In this study, we propose a framework for understanding DLK mediated neuronal cytoskeletal homeostasis. We demonstrate that low dose nocodazole treatment activates DLK signaling. Activation of DLK signaling results in a DLK-dependent transcriptional signature, which we identify through RNA-seq. This signature includes genes likely to attenuate DLK signaling while simultaneously inducing actin regulating genes. We identify alterations to the cytoskeleton including actin-based morphological changes to the axon. These results are consistent with the model that cytoskeletal disruption in the neuron induces a DLK-dependent homeostatic mechanism, which we term the Cytoskeletal Stress Response (CSR) pathway.

Genome-wide analysis and expression profiling of the HD-ZIP gene family in kiwifruit.

The homeodomain-leucine zipper (HD-Zip) gene family plays a pivotal role in plant development and stress responses. Nevertheless, a comprehensive characterization of the HD-Zip gene family in kiwifruit has been lacking. In this study, we have systematically identified 70 HD-Zip genes in the Actinidia chinensis (Ac) genome and 55 in the Actinidia eriantha (Ae) genome. These genes have been categorized into four subfamilies (HD-Zip I, II, III, and IV) through rigorous phylogenetic analysis. Analysis of synteny patterns and selection pressures has provided insights into how whole-genome duplication (WGD) or segmental may have contributed to the divergence in gene numbers between these two kiwifruit species, with duplicated gene pairs undergoing purifying selection. Furthermore, our study has unveiled tissue-specific expression patterns among kiwifruit HD-Zip genes, with some genes identified as key regulators of kiwifruit responses to bacterial canker disease and postharvest processes. These findings not only offer valuable insights into the evolutionary and functional characteristics of kiwifruit HD-Zips but also shed light on their potential roles in plant growth and development.

Leucine Zipper Downregulated in Cancer-1 Interacts with Clathrin Adaptors to Control Epidermal Growth Factor Receptor (EGFR) Internalization and Gefitinib Response in EGFR-Mutated Non-Small Cell Lung Cancer.

The epidermal growth factor receptor (EGFR) is a common driver of non-small cell lung cancer (NSCLC). Clathrin-mediated internalization (CMI) sustains EGFR signaling. AXL is associated with resistance to EGFR-tyrosine kinase inhibitors (TKIs) in EGFR-mutated (EGFRM) NSCLC. We investigated the effects of Leucine zipper downregulated in cancer-1 (LDOC1) on EGFR CMI and NSCLC treatment. Coimmunoprecipitation, double immunofluorescence staining, confocal microscopy analysis, cell surface labelling assays, and immunohistochemistry studies were conducted. We revealed that LDOC1 interacts with clathrin adaptors through binding motifs. LDOC1 depletion promotes internalization and plasma membrane recycling of EGFR in EGFRM NSCLC PC9 and HCC827 cells. Membranous and cytoplasmic EGFR decreased and increased, respectively, in LDOC1 (-) NSCLC tumors. LDOC1 depletion enhanced and sustained activation of EGFR, AXL, and HER2 and enhanced activation of HER3 in PC9 and HCC827 cells. Sensitivity to first-generation EGFR-TKIs (gefitinib and erlotinib) was significantly reduced in LDOC1-depleted PC9 and HCC827 cells. Moreover, LDOC1 downregulation was significantly associated (p < 0.001) with poor overall survival in patients with EGFRM NSCLC receiving gefitinib (n = 100). In conclusion, LDOC1 may regulate the efficacy of first-generation EGFR-TKIs by participating in the CMI of EGFR. Accordingly, LDOC1 may function as a prognostic biomarker for EGFRM NSCLC.

Regulation of the Activity of the Dual Leucine Zipper Kinase by Distinct Mechanisms.

The dual leucine zipper kinase (DLK) alias mitogen-activated protein 3 kinase 12 (MAP3K12) has gained much attention in recent years. DLK belongs to the mixed lineage kinases, characterized by homology to serine/threonine and tyrosine kinase, but exerts serine/threonine kinase activity. DLK has been implicated in many diseases, including several neurodegenerative diseases, glaucoma, and diabetes mellitus. As a MAP3K, it is generally assumed that DLK becomes phosphorylated and activated by upstream signals and phosphorylates and activates itself, the downstream serine/threonine MAP2K, and, ultimately, MAPK. In addition, other mechanisms such as protein-protein interactions, proteasomal degradation, dephosphorylation by various phosphatases, palmitoylation, and subcellular localization have been shown to be involved in the regulation of DLK activity or its fine-tuning. In the present review, the diverse mechanisms regulating DLK activity will be summarized to provide better insights into DLK action and, possibly, new targets to modulate DLK function.

Comprehensive bioinformation analysis of homeodomain-leucine zipper gene family and expression pattern of HD-Zip I under abiotic stress in Salix suchowensis.

BACKGROUND: Homeodomain-leucine zipper (HD-Zip) transcription factors are plant-specific and play important roles in plant defense against environmental stresses. Identification and functional studies have been carried out in model plants such as rice, Arabidopsis thaliana, and poplar, but comprehensive analysis on the HD-Zip family of Salix suchowensis have not been reported. RESULTS: A total of 55 HD-Zip genes were identified in the willow genome, unevenly distributed on 18 chromosomes except for chromosome 19. And segmental duplication events containing SsHD-Zip were detected on all chromosomes except chromosomes 13 and 19. The SsHD-Zip were classified into 4 subfamilies subfamilies (I-IV) according to the evolutionary analysis, and members of each subfamily shared similar domain structure and gene structure. The combination of GO annotation and promoter analysis showed that SsHD-Zip genes responded to multiple abiotic stresses. Furthermore, the results of qPCR analysis showed that the SsHD-Zip I gene exhibited different degrees of expression under salt stress, PEG treatment and heat treatment. Moreover, there was a synergistic effect between SsHD-Zip I genes under stress conditions based on coregulatory networks analysis. CONCLUSIONS: In this study, HD-Zip transcription factors were systematically identified and analyzed at the whole genome level. These results preliminarily clarified the structural characteristics and related functions of willow HD-Zip family members, and it was found that SsHox34, SsHox36 and SsHox51 genes were significantly involved in the response to various stresses. Together, these findings laid the foundation for further research on the resistance functions of willow HD-Zip genes.

Overexpression of JNK-associated leucine zipper protein induces chromosomal instability through interaction with dynein light intermediate chain 1.

The c-Jun N-terminal kinase-associated leucine zipper protein (JLP), a scaffold protein of mitogen-activated protein kinase signaling pathways, is a multifunctional protein involved in a variety of cellular processes. It has been reported that JLP is overexpressed in various types of cancer and is expected to be a potential therapeutic target. However, whether and how JLP overexpression affects non-transformed cells remain unknown. Here, we aimed to investigate the effect of JLP overexpression on chromosomal stability in human non-transformed cells and the mechanisms involved. We found that aneuploidy was induced in JLP-overexpressed cells. Moreover, we established JLP-inducible cell lines and observed an increased frequency of chromosome missegregation, reduced time from nuclear envelope breakdown to anaphase onset, and decreased levels of the spindle assembly checkpoint (SAC) components at the prometaphase kinetochore in cells overexpressing the wild-type JLP. In contrast, we observed that a point mutant JLP lacking the ability to interact with dynein light intermediate chain 1 (DLIC1) failed to induce chromosomal instability. Our results suggest that overexpression of the wild-type JLP facilitates premature SAC silencing through interaction with DLIC1, leading to aneuploidy. This study provides a novel insight into the mechanism through which JLP overexpression is associated with cancer development and progression.

Leucine zipper protein 1 (LUZP1) regulates the constriction velocity of the contractile ring during cytokinesis.

There has been a great deal of research on cell division and its mechanisms; however, its processes still have many unknowns. To find novel proteins that regulate cell division, we performed the screening using siRNAs and/or the expression plasmid of the target genes and identified leucine zipper protein 1 (LUZP1). Recent studies have shown that LUZP1 interacts with various proteins and stabilizes the actin cytoskeleton; however, the function of LUZP1 in mitosis is not known. In this study, we found that LUZP1 colocalized with the chromosomal passenger complex (CPC) at the centromere in metaphase and at the central spindle in anaphase and that these LUZP1 localizations were regulated by CPC activity and kinesin family member 20A (KIF20A). Mass spectrometry analysis identified that LUZP1 interacted with death-associated protein kinase 3 (DAPK3), one regulator of the cleavage furrow ingression in cytokinesis. In addition, we found that LUZP1 also interacted with myosin light chain 9 (MYL9), a substrate of DAPK3, and comprehensively inhibited MYL9 phosphorylation by DAPK3. In line with a known role for MYL9 in the actin-myosin contraction, LUZP1 suppression accelerated the constriction velocity at the division plane in our time-lapse analysis. Our study indicates that LUZP1 is a novel regulator for cytokinesis that regulates the constriction velocity of the contractile ring.

TGFß1-driven SMAD2/3 phosphorylation and myofibroblast emergence are fully dependent on the TGFß1 pre-activation of MAPKs and controlled by maternal leucine zipper kinase.

Following wounding, endogenously secreted TGFßs drive resident and bone marrow-derived cells to convert into α-smooth actin (SMA)-rich, contractile myofibroblasts. The TGFß effect is initiated by the phosphorylation of SMADs 2 and 3 (SMAD2/3). This event has been referred to as the canonical response to TGFß. TGFß also elicits other responses viewed as parallel events not directly connected to the SMAD activation, and thus referred to as noncanonical. A recognized response is the phosphorylation of the -activated kinase (TAK1/MAP3K), an upstream component of the mitogen-activated protein kinase (MAPK) cascade. We have now examined the relationship between these two effects of TGFß1 at their earliest stages. The bulk of the studies were carried out with primary fibroblasts derived from the human cornea. The results' widespread relevance was confirmed in critical experiments with dermal-, and Tenon's capsule-derived fibroblasts. Cells were treated with kinase inhibitors or targeting siRNAs followed by induction by 2 ng/ml TGFß1, and/or 10 ng/ml TNF-α. Cells were collected after 1 to 30 min for Western blot analysis and assayed for the accumulation of phosphorylated TAK1, ASK1, JNK1/2, p38, HPS27, MELK, SMAD2/3, and GAPDH. The effect of the kinase inhibitors on α-SMA expression and α-SMA stress fiber organization was also tested. For the immediate response to TGFß1 we found that a) activation of the MAPK pathway was completed within 1 min after the addition of TGFß1; b) phosphorylation of JNK1/2 was fully dependent on TAK1 and ASK1 activity, c) phosphorylation of MELK was fully dependent on JNK1/2 activity; d) phosphorylation of ASK1 depends on MELK activity, indicating the existence of an ASK1-MELK positive activation feedback loop; e) phosphorylation of SMAD2/3 started only after a 5 min period and reached a nadir after 10-15 min, f) the latter phosphorylation was fully blocked by inhibition of TAK1, ASK1, JNK1/2, and MELK, and siRNA-driven MELK downregulation; g) the inhibitors equally blocked the α-SMA protein expression, stress fiber development, and cell morphology changes at 72 h. These results demonstrate that the activation of the canonical pathway is fully subordinate to the activity of the MAPK pathway, challenging the concept of canonical and noncanonical TGFß pathways and that SMAD2/3 activation is mediated by MELK, a kinase not previously associated with rapid pharmacological responses.

PrLZ regulates EMT and invasion in prostate cancer via the TGF-ß1/p-smad2/miR-200 family/ZEB1 axis.

BACKGROUND: Prostate leucine zipper (PrLZ) is a prostate-specific protein, and our previous study demonstrated that PrLZ enhances the malignant progression of prostate cancer (Pca). However, the roles of PrLZ in epithelial to mesenchymal transition (EMT) remain unknown. METHODS: Quantitative real-time PCR (qRT-PCR), immunohistochemical (IHC) staining, hematoxylin-eosin (HE) staining, and western blotting were used to analyze the expression of protein and genes level in human PCa cell lines. Invasion assay was used to examine the effect of PrLZ, miR-200a, miR-200b, miR-200c, miR-141, miR-429, miR-205, and ZEB1 on PCa cell line invasion in vitro. Prostate cancer metastasis animal model was designed to assess the effect of PrLZ on PCa cell line invasion in vivo. RESULTS: We proved that high PrLZ expression initiates EMT, which was shown by the downregulation of E-cadherin and upregulation of vimentin in PC-3/PrLZ and ARCaP-E/PrLZ cells. Mechanistic analysis revealed that PrLZ regulates EMT by activating TGF-ß1/p-smad2 signaling and further inhibiting the expression of miR-200 family members, which negatively regulates ZEB1 expression and causes EMT in Pca. Moreover, using two of orthotopic mouse model and tail vein injection of human prostate cancer cells mouse model, we observed that PC-3/PrLZ cells led to the development of distant organ metastases in vivo. CONCLUSIONS: Our results show the mechanism by which PrLZ regulates EMT and metastasis and suggest that PrLZ may be a potential therapeutic target for Pca metastasis.

Effects of acute exercise and training status on glucocorticoid-induced leucine zipper (GILZ) expression in human skeletal muscle.

Interactions between statin therapy and physical exercise complicate effective cardiovascular prevention. Emerging evidence suggests that muscle strain related changes in the expression of the glucocorticoid-induced leucine zipper (GILZ) may be involved. Therefore, we measured GILZ mRNA expression levels in M. vastus lateralis samples of 32 healthy individuals before and after a standardized bout of strength or endurance exercise. Overall, we found a highly significant downregulation of GILZ after exercise training (p < 0.001). Within-subgroup changes were statistically significant only after strength training, supporting the role of muscle (as opposed to cardiocirculatory) strain. If confirmed, this may help fitting training recommendations and medication.

The Homeodomain-Leucine Zipper Subfamily I Contributes to Leaf Age- and Time-Dependent Resistance to Pathogens in Arabidopsis thaliana.

In Arabidopsis thaliana (Arabidopsis), nonhost resistance (NHR) is influenced by both leaf age and the moment of inoculation. While the circadian clock and photoperiod have been linked to the time-dependent regulation of NHR in Arabidopsis, the mechanism underlying leaf age-dependent NHR remains unclear. In this study, we investigated leaf age-dependent NHR to Pyricularia oryzae in Arabidopsis. Our findings revealed that this NHR type is regulated by both miR156-dependent and miR156-independent pathways. To identify the key players, we utilized rice-FOX Arabidopsis lines and identified the rice HD-Zip I OsHOX6 gene. Notably, OsHOX6 expression confers robust NHR to P. oryzae and Colletotrichum nymphaeae in Arabidopsis, with its effect being contingent upon leaf age. Moreover, we explored the role of AtHB7 and AtHB12, the Arabidopsis closest homologues of OsHOX6, by studying mutants and overexpressors in Arabidopsis-C. higginsianum interaction. AtHB7 and AtHB12 were found to contribute to both penetration resistance and post-penetration resistance to C. higginsianum in a leaf age- and time-dependent manner. These findings highlight the involvement of HD-Zip I AtHB7 and AtHB12, well-known regulators of development and abiotic stress responses, in biotic stress responses in Arabidopsis.

The leucine zipper domain of the transcriptional repressor Opi1 underlies a signal transduction mechanism regulating lipid synthesis.

In Saccharomyces cerevisiae, the transcriptional repressor Opi1 regulates the expression of genes involved in phospholipid synthesis responding to the abundance of the phospholipid precursor phosphatidic acid at the endoplasmic reticulum. We report here the identification of the conserved leucine zipper (LZ) domain of Opi1 as a hot spot for gain of function mutations and the characterization of the strongest variant identified, Opi1N150D. LZ modeling posits asparagine 150 embedded on the hydrophobic surface of the zipper and specifying dynamic parallel homodimerization by allowing electrostatic bonding across the hydrophobic dimerization interface. Opi1 variants carrying any of the other three ionic residues at amino acid 150 were also repressing. Genetic analyses showed that Opi1N150D variant is dominant, and its phenotype is attenuated when loss of function mutations identified in the other two conserved domains are present in cis. We build on the notion that membrane binding facilitates LZ dimerization to antagonize an intramolecular interaction of the zipper necessary for repression. Dissecting Opi1 protein in three polypeptides containing each conserved region, we performed in vitro analyses to explore interdomain interactions. An Opi11-190 probe interacted with Opi1291-404, the C terminus that bears the activator interacting domain (AID). LZ or AID loss of function mutations attenuated the interaction of the probes but was unaffected by the N150D mutation. We propose a model for Opi1 signal transduction whereby synergy between membrane-binding events and LZ dimerization antagonizes intramolecular LZ-AID interaction and transcriptional repression.

HIV-1 reverse transcriptase stability correlates with Gag cleavage efficiency: reverse transcriptase interaction implications for modulating protease activation.

Proteolytic processing of human immunodeficiency virus type 1 particles mediated by viral protease (PR) is essential for acquiring virus infectivity. Activation of PR embedded in Gag-Pol is triggered by Gag-Pol dimerization during virus assembly. We previously reported that amino acid substitutions at the RT tryptophan repeat motif destabilize virus-associated RT and attenuate the ability of efavirenz (EFV, an RT dimerization enhancer) to increase PR-mediated Gag cleavage efficiency. Furthermore, a single amino acid change at RT significantly reduces virus yields due to enhanced Gag cleavage. These data raise the possibility of the RT domain contributing to PR activation by promoting Gag-Pol dimerization. To test this hypothesis, we investigated the putative involvement of a hydrophobic leucine repeat motif (LRM) spanning RT L282 to L310 in RT/RT interactions. We found that LRM amino acid substitutions led to RT instability and that RT is consequently susceptible to degradation by PR. The LRM mutants exhibited reduced Gag cleavage efficiencies while attenuating the EFV enhancement of Gag cleavage. In addition, an RT dimerization-defective mutant, W401A, reduced enhanced Gag cleavage via a leucine zipper (LZ) motif inserted at the deleted Gag-Pol region. Importantly, the presence of RT and integrase domains failed to counteract the LZ enhancement of Gag cleavage. A combination of the Gag cleavage enhancement factors EFV and W402A markedly impaired Gag cleavage, indicating a disruption of W402A Gag-Pol dimerization following EFV binding to W402A Gag-Pol. Our results support the idea that RT modulates PR activation by affecting Gag-Pol/Gag-Pol interaction. IMPORTANCE A stable reverse transcriptase (RT) p66/51 heterodimer is required for HIV-1 genome replication in host cells following virus entry. The activation of viral protease (PR) to mediate virus particle processing helps viruses acquire infectivity following cell release. RT and PR both appear to be major targets for inhibiting HIV-1 replication. We found a strong correlation between impaired p66/51RT stability and deficient PR-mediated Gag cleavage, suggesting that RT/RT interaction is critical for triggering PR activation via the promotion of adequate Gag-Pol dimerization. Accordingly, RT/RT interaction is a potentially advantageous method for anti-HIV/AIDS therapy if it is found to simultaneously block PR and RT enzymatic activity.

Discovering immunoreceptor coupling and organization motifs.

The recently determined cryo-EM structures of the T cell antigen receptor (TCR) and B cell antigen receptor (BCR) show in molecular details the interactions of the ligand-binding part with the signaling subunits but they do not reveal the signaling mechanism of these antigen receptors. Without knowing the molecular basis of antigen sensing by these receptors, a rational design of optimal vaccines is not possible. The existence of conserved amino acids (AAs) that are not involved in the subunit interaction suggests that antigen receptors form higher complexes and/or have lateral interactors that control their activity. Here, I describe evolutionary conserved leucine zipper (LZ) motifs within the transmembrane domains (TMD) of antigen and coreceptor components that are likely to be involved in the oligomerization and lateral interaction of antigen receptor complexes on T and B cells. These immunoreceptor coupling and organization motifs (ICOMs) are also found within the TMDs of other important receptor types and viral envelope proteins. This discovery suggests that antigen receptors do not function as isolated entities but rather as part of an ICOM-based interactome that controls their nanoscale organization on resting cells and their dynamic remodeling on activated lymphocytes.

In-vitro anticancer evaluation of newly designed and characterized tri/tetra-substituted imidazole congeners- maternal embryonic leucine zipper kinase inhibitors: Molecular docking and MD simulation approaches.

Our cascading attempt to develop new potent molecules now involves designing a series of imidazole derivatives and synthesizing two sets of 2,4,5- tri-substituted (4a-4d) and 1,2,4,5-tetra-substituted (6a-6d) imidazole by the principle of Debus-Radziszewski multicomponent synthesis reaction. The structures of the obtained compounds were confirmed by 1H/13C NMR, FT-IR, elemental analysis, purity and the retention time was analyzed by HPLC. Based upon the binding affinity in the molecular docking studies, we have synthesized different imidazole derivatives from which compound 6c have been found to show more anti-proliferative activity by inducing apoptosis at a higher rate than the other compounds corroborating the in-silico prediction. The structure and crystallinity of compound 4d have been confirmed by single XRD analysis. The synthesized molecules were screened for their in vitro anti-cancer properties in triple negative breast cancer cell line (MDA-MB-231), pancreatic cancer cell lines (MIA PaCa-2) and oral squamous cell carcinoma cell line (H357) and results indicated that all the compounds inhibited the cell proliferation in a concentration-dependent manner at different time points. The compounds 4b and 6d were found to be effective against the S. aureus bacterial strain whereas only compound 4d fairly inhibited the fungal strain of T. rubrum with a MIC 12.5 µg/mL. Molecular docking study reveals good interaction of the synthesized compounds with known target MELK involved in oncogenesis having high binding profiles. The lead compound 6c was further analyzed by the detailed molecular dynamics study to establish the stability of the ligand-enzyme complex.

An Encodable Scaffold for Sequence-Specific Recognition of Duplex RNA.

RNA, unlike DNA, folds into a multitude of secondary and tertiary structures. This structural diversity has impeded the development of ligands that can sequence-specifically target this biomolecule. We sought to develop ligands for double-stranded RNA (dsRNA) segments, which are ubiquitous in RNA tertiary structure. The major groove of double-stranded DNA is sequence-specifically recognized by a range of dimeric helical transcription factors, including the basic leucine zippers (bZIP) and basic helix-loop-helix (bHLH) proteins; however, such simple structural motifs are not prevalent in RNA-binding proteins. We interrogated the high-resolution structures of DNA and RNA to identify requirements for a helix fork motif to occupy dsRNA major grooves akin to dsDNA. Our analysis suggested that the rigidity and angle of approach of dimeric helices in bZIP/bHLH motifs are not ideal for the binding of dsRNA major grooves. This investigation revealed that the replacement of the leucine zipper motifs in bHLH proteins with synthetic crosslinkers would allow recognition of dsRNA. We show that a model bHLH DNA-binding motif does not bind dsRNA but can be reengineered as an RNA ligand. Based on this hypothesis, we rationally designed a miniature synthetic crosslinked helix fork (CHF) as a generalizable proteomimetic scaffold for targeting dsRNA. We evaluated several CHF constructs against a set of RNA and DNA hairpins to probe the specificity of the designed construct. Our studies reveal a new class of proteomimetics as an encodable platform for sequence-specific recognition of dsRNA.

Cooperation between bHLH transcription factors and histones for DNA access.

The basic helix-loop-helix (bHLH) family of transcription factors recognizes DNA motifs known as E-boxes (CANNTG) and includes 108 members1. Here we investigate how chromatinized E-boxes are engaged by two structurally diverse bHLH proteins: the proto-oncogene MYC-MAX and the circadian transcription factor CLOCK-BMAL1 (refs. 2,3). Both transcription factors bind to E-boxes preferentially near the nucleosomal entry-exit sites. Structural studies with engineered or native nucleosome sequences show that MYC-MAX or CLOCK-BMAL1 triggers the release of DNA from histones to gain access. Atop the H2A-H2B acidic patch4, the CLOCK-BMAL1 Per-Arnt-Sim (PAS) dimerization domains engage the histone octamer disc. Binding of tandem E-boxes5-7 at endogenous DNA sequences occurs through direct interactions between two CLOCK-BMAL1 protomers and histones and is important for circadian cycling. At internal E-boxes, the MYC-MAX leucine zipper can also interact with histones H2B and H3, and its binding is indirectly enhanced by OCT4 elsewhere on the nucleosome. The nucleosomal E-box position and the type of bHLH dimerization domain jointly determine the histone contact, the affinity and the degree of competition and cooperativity with other nucleosome-bound factors.