Dimerization choice and alternative functions of ZBTB transcription factors.

Zinc Finger DNA-binding domain-containing proteins are the most populous family among eukaryotic transcription factors. Among these, members of the BTB domain-containing ZBTB sub-family are mostly known for their transcriptional repressive functions. In this Viewpoint article, we explore molecular mechanisms that potentially diversify the function of ZBTB proteins based on their homo and heterodimerization, alternative splicing and post-translational modifications. We describe how the BTB domain is as much a scaffold for the assembly of co-repressors, as a domain that regulates protein stability. We highlight another mechanism that regulates ZBTB protein stability: phosphorylation in the zinc finger domain. We explore the non-transcriptional, structural roles of ZBTB proteins and highlight novel findings that describe the ability of ZBTB proteins to associate with poly adenosine ribose in the nucleus during the DNA damage response. Herein, we discuss the contribution of BTB domain scaffolds to the formation of transcriptional repressive complexes, to chromosome compartmentalization and their non-transcriptional, purely structural functions in the nucleus.

Evaluation of Clinical and Immunological Alterations Associated with ICF Syndrome.

PURPOSE: Immunodeficiency with centromeric instability and facial anomalies (ICF) syndrome is a rare autosomal recessive combined immunodeficiency. The detailed immune responses are not explored widely. We investigated known and novel immune alterations in lymphocyte subpopulations and their association with clinical symptoms in a well-defined ICF cohort. METHODS: We recruited the clinical findings from twelve ICF1 and ICF2 patients. We performed detailed immunological evaluation, including lymphocyte subset analyses, upregulation, and proliferation of T cells. We also determined the frequency of circulating T follicular helper (cTFH) and regulatory T (Treg) cells and their subtypes by flow cytometry. RESULTS: There were ten ICF1 and two ICF2 patients. We identified two novel homozygous missense mutations in the ZBTB24 gene. Respiratory tract infections were the most common recurrent infections among the patients. Gastrointestinal system (GIS) involvements were observed in seven patients. All patients received intravenous immunoglobulin replacement therapy and antibacterial prophylaxis; two died during the follow-up period. Immunologically, CD4+ T-cell counts, percentages of recent thymic emigrant T cells, and naive CD4+ T decreased in two, five, and four patients, respectively. Impaired T-cell proliferation and reduced CD25 upregulation were detected in all patients. These changes were more prominent in CD8+ T cells. GIS involvements negatively correlated with CD3+ T-, CD3+CD4+ T-, CD16+CD56+ NK-cell counts, and CD4+/CD8+ T-cell ratios. Further, we observed expanded cTFH cells and reduced Treg and follicular regulatory T cells with a skewing to a TH2-like phenotype in all tested subpopulations. CONCLUSION: The ICF syndrome encompasses various manifestations affecting multiple end organs. Perturbed T-cell responses with increased cTFH and decreased Treg cells may provide further insight into the immune aberrations observed in ICF syndrome.

Human ACE2 orthologous peptide sequences show better binding affinity to SARS-CoV-2 RBD domain: Implications for drug design.

Computational methods coupled with experimental validation play a critical role in the identification of novel inhibitory peptides that interact with viral antigenic determinants. The interaction between the receptor binding domain (RBD) of SARS-CoV-2 spike protein and the helical peptide of human angiotensin-converting enzyme-2 (ACE2) is a necessity for the initiation of viral infection. Herein, natural orthologs of human ACE2 helical peptide were evaluated for competitive inhibitory binding to the viral RBD by use of a computational approach, which was experimentally validated. A total of 624 natural ACE2 orthologous 32-amino acid long peptides were identified through a similarity search. Molecular docking was used to virtually screen and rank the peptides based on binding affinity metrics, benchmarked against human ACE2 peptide docked to the RBD. Molecular dynamics (MD) simulations were done for the human reference and the Nipponia nippon peptide as it exhibited the highest binding affinity (Gibbs free energy; -14 kcal/mol) predicted from the docking results. The MD simulation confirmed the stability of the assessed peptide in the complex (-12.3 kcal/mol). The top three docked-peptides (from Chitinophaga sancti, Nipponia nippon, and Mus musculus) and the human reference were experimentally validated by use of surface plasmon resonance technology. The human reference exhibited the weakest binding affinity (Kd of 318-441 pM) among the peptides tested, in agreement with the docking prediction, while the peptide from Nipponia nippon was the best, with 267-538-fold higher affinity than the reference. The validated peptides merit further investigation. This work showcases that the approach herein can aid in the identification of inhibitory biosimilar peptides for other viruses.

Expression and characterization of recombinant IL-1Ra in Aspergillus oryzae as a system.

BACKGROUND: The interleukin-1 receptor antagonist (IL-1Ra) is a crucial molecule that counteracts the effects of interleukin-1 (IL-1) by binding to its receptor. A high concentration of IL-1Ra is required for complete inhibition of IL-1 activity. However, the currently available Escherichia coli-expressed IL-1Ra (E. coli IL-1Ra, Anakinra) has a limited half-life. This study aims to produce a cost-effective, functional IL-1Ra on an industrial scale by expressing it in the pyrG auxotroph Aspergillus oryzae. RESULTS: We purified A. oryzae-expressed IL-1Ra (Asp. IL-1Ra) using ion exchange and size exclusion chromatography (53 mg/L). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that Asp. IL-1Ra is N-glycosylated and approximately 17 kDa in size. We conducted a comparative study of the bioactivity, binding kinetics, and half-life between Asp. IL-1Ra and E. coli IL-1Ra. Asp. IL-1Ra showed good bioactivity even at a low concentration of 0.5 nM. The in vitro half-life of Asp. IL-1Ra was determined for different time points (0, 24, 48, 72, and 96 h) and showed higher stability than E. coli IL-1Ra, despite exhibiting a 100-fold lower binding affinity (2 nM). CONCLUSION: This study reports the production of a functional Asp. IL-1Ra with advantageous stability, without extensive downstream processing. To our knowledge, this is the first report of a recombinant functional and stable IL-1Ra expressed in A. oryzae. Our results suggest that Asp. IL-1Ra has potential for industrial-scale production as a cost-effective alternative to E. coli IL-1Ra.

Sibling rivalry among the ZBTB transcription factor family: homodimers versus heterodimers.

The BTB domain is an oligomerization domain found in over 300 proteins encoded in the human genome. In the family of BTB domain and zinc finger-containing (ZBTB) transcription factors, 49 members share the same protein architecture. The N-terminal BTB domain is structurally conserved among the family members and serves as the dimerization site, whereas the C-terminal zinc finger motifs mediate DNA binding. The available BTB domain structures from this family reveal a natural inclination for homodimerization. In this study, we investigated the potential for heterodimer formation in the cellular environment. We selected five BTB homodimers and four heterodimer structures. We performed cell-based binding assays with fluorescent protein-BTB domain fusions to assess dimer formation. We tested the binding of several BTB pairs, and we were able to confirm the heterodimeric physical interaction between the BTB domains of PATZ1 and PATZ2, previously reported only in an interactome mapping experiment. We also found this pair to be co-expressed in several immune system cell types. Finally, we used the available structures of BTB domain dimers and newly constructed models in extended molecular dynamics simulations (500 ns) to understand the energetic determinants of homo- and heterodimer formation. We conclude that heterodimer formation, although frequently described as less preferred than homodimers, is a possible mechanism to increase the combinatorial specificity of this transcription factor family.

Targeting mitochondrial DNA polymerase gamma for selective inhibition of MLH1 deficient colon cancer growth.

Synthetic lethality in DNA repair pathways is an important strategy for the selective treatment of cancer cells without harming healthy cells and developing cancer-specific drugs. The synthetic lethal interaction between the mismatch repair (MMR) protein, MutL homolog 1 (MLH1), and the mitochondrial base excision repair protein, DNA polymerase γ (Pol γ) was used in this study for the selective treatment of MLH1 deficient cancers. Germline mutations in the MLH1 gene and aberrant MLH1 promoter methylation result in an increased risk of developing many cancers, including nonpolyposis colorectal and endometrial cancers. Because the inhibition of Pol γ in MLH1 deficient cancer cells provides the synthetic lethal selectivity, we conducted a comprehensive small molecule screening from various databases and chemical drug library molecules for novel Pol γ inhibitors that selectively kill MLH1 deficient cancer cells. We characterized these Pol γ inhibitor molecules in vitro and in vivo, and identified 3,3'-[(1,1'-Biphenyl)-4',4'-diyl)bis(azo)]bis[4-amino-1-naphthalenesulfonic acid] (congo red; CR; Zinc 03830554) as a high-affinity binder to the Pol γ protein and potent inhibitor of the Pol γ strand displacement and one-nucleotide incorporation DNA synthesis activities in vitro and in vivo. CR reduced the cell proliferation of MLH1 deficient HCT116 human colon cancer cells and suppressed HCT116 xenograft tumor growth whereas it did not affect the MLH1 proficient cell proliferation and xenograft tumor growth. CR caused mitochondrial dysfunction and cell death by inhibiting Pol γ activity and oxidative mtDNA damage repair, increasing the production of reactive oxygen species and oxidative mtDNA damage in MLH1 deficient cells. This study suggests that the Pol γ inhibitor, CR may be further evaluated for the MLH1 deficient cancers' therapy.

Reversal of the T cell immune system reveals the molecular basis for T cell lineage fate determination in the thymus.

T cell specificity and function are linked during development, as MHC-II-specific TCR signals generate CD4 helper T cells and MHC-I-specific TCR signals generate CD8 cytotoxic T cells, but the basis remains uncertain. We now report that switching coreceptor proteins encoded by Cd4 and Cd8 gene loci functionally reverses the T cell immune system, generating CD4 cytotoxic and CD8 helper T cells. Such functional reversal reveals that coreceptor proteins promote the helper-lineage fate when encoded by Cd4, but promote the cytotoxic-lineage fate when encoded in Cd8-regardless of the coreceptor proteins each locus encodes. Thus, T cell lineage fate is determined by cis-regulatory elements in coreceptor gene loci and is not determined by the coreceptor proteins they encode, invalidating coreceptor signal strength as the basis of lineage fate determination. Moreover, we consider that evolution selected the particular coreceptor proteins that Cd4 and Cd8 gene loci encode to avoid generating functionally reversed T cells because they fail to promote protective immunity against environmental pathogens.

Nanobodies as molecular imaging probes.

Camelidae derived single-domain antibodies (sdAbs), commonly known as nanobodies (Nbs), are the smallest antibody fragments with full antigen-binding capacity. Owing to their desirable properties such as small size, high specificity, strong affinity, excellent stability, and modularity, nanobodies are on their way to overtake conventional antibodies in terms of popularity. To date, a broad range of nanobodies have been generated against different molecular targets with applications spanning basic research, diagnostics, and therapeutics. In the field of molecular imaging, nanobody-based probes have emerged as a powerful tool. Radioactive or fluorescently labeled nanobodies are now used to detect and track many targets in different biological systems using imaging techniques. In this review, we provide an overview of the use of nanobodies as molecular probes. Additionally, we discuss current techniques for the generation, conjugation, and intracellular delivery of nanobodies.

Synchronous and Asynchronous Response in Dynamically Perturbed Proteins.

We present a dynamic perturbation-response model of proteins based on the Gaussian Network Model, where a residue is perturbed periodically, and the dynamic response of other residues is determined. The model shows that periodic perturbation causes a synchronous response in phase with the perturbation and an asynchronous response that is out of phase. The asynchronous component results from the viscous effects of the solvent and other dispersive factors in the system. The model is based on the solution of the Langevin equation in the presence of solvent, noise, and perturbation. We introduce several novel ideas: The concept of storage and loss compliance of the protein and their dependence on structure and frequency; the amount of work lost and the residues that contribute significantly to the lost work; new dynamic correlations that result from perturbation; causality, that is, the response of j when i is perturbed is not equal to the response of i when j is perturbed. As examples, we study two systems, namely, bovine rhodopsin and the class of nanobodies. The general results obtained are (i) synchronous and asynchronous correlations depend strongly on the frequency of perturbation, their magnitude decreases with increasing frequency, (ii) time-delayed mean-squared fluctuations of residues have only synchronous components. Asynchronicity is present only in cross correlations, that is, correlations between different residues, (iii) perturbation of loop residues leads to a large dissipation of work, (iv) correlations satisfy the hypothesis of pre-existing pathways according to which information transfer by perturbation rides on already existing equilibrium correlations in the system, (v) dynamic perturbation can introduce a selective response in the system, where the perturbation of each residue excites different sets of responding residues, and (vi) it is possible to identify nondissipative residues whose perturbation does not lead to dissipation in the protein. Despite its simplicity, the model explains several features of allosteric manipulation.

Structural analysis of the PATZ1 BTB domain homodimer.

PATZ1 is a ubiquitously expressed transcriptional repressor belonging to the ZBTB family that is functionally expressed in T lymphocytes. PATZ1 targets the CD8 gene in lymphocyte development and interacts with the p53 protein to control genes that are important in proliferation and in the DNA-damage response. PATZ1 exerts its activity through an N-terminal BTB domain that mediates dimerization and co-repressor interactions and a C-terminal zinc-finger motif-containing domain that mediates DNA binding. Here, the crystal structures of the murine and zebrafish PATZ1 BTB domains are reported at 2.3 and 1.8 Šresolution, respectively. The structures revealed that the PATZ1 BTB domain forms a stable homodimer with a lateral surface groove, as in other ZBTB structures. Analysis of the lateral groove revealed a large acidic patch in this region, which contrasts with the previously resolved basic co-repressor binding interface of BCL6. A large 30-amino-acid glycine- and alanine-rich central loop, which is unique to mammalian PATZ1 amongst all ZBTB proteins, could not be resolved, probably owing to its flexibility. Molecular-dynamics simulations suggest a contribution of this loop to modulation of the mammalian BTB dimerization interface.

Doxorubicin inhibits miR-140 expression and upregulates PD-L1 expression in HCT116 cells, opposite to its effects on MDA-MB-231 cells.

One of the most challenging problems in colorectal cancer (CRC) is resistance to chemotherapy drugs such as doxorubicin (DOX). The programmed death ligand-1 (PD-L1) is related to chemoresistance and is overexpressed in several human cancer cell types. Here, we investigated the changes in the expression of PD-L1 in DOX-treated CRC and breast cancer (BRC) cells. Also, to address PD-L1 regulation, we assessed expression levels of miR-140 and miR-34a, two microRNAs that can target the 3' UTR region of the gene encoding PD-L1. HCT116 CRC and MDA-MB-231 BRC cells were treated with various doses of DOX in culture and PD-L1 expression was quantified using qRT-PCR, flow cytometry, and western blot analysis. We also evaluated PD-L1 localization in HCT116 cells by immunofluorescence. Next, we assessed expression of miR-140 and miR-34a in DOX-treated HCT116 and MDA-MB-231 cells. Finally, we investigated whether miR-140 targets the 3' UTR of the gene encoding PD-L1 in HCT116 cells using the p2FP-RNAi RNAi reporter vector system. PD-L1 expression in HCT116 cells, while low at baseline, can be induced by treatment with 0.5 µM DOX. MDA-MB-231 baseline PD-L1 expression exceeded HCT116 cell maximal expression and decreased following DOX treatment. We further demonstrated that PD-L1 localizes to the cell surface in DOX-treated HCT116 cells. While miR-140 expression decreased in DOX-treated HCT116 cells, it increased in DOX-treated MDA-MB-231 cells. MiR-34a expression increased in both DOX-treated cell types. Finally, we present evidence for the regulation of PD-L1 by miR-140 in HCT116 cells. PD-L1 expression can increase following treatment with DOX in HCT116 cells but decrease in MDA-MB-231 cells, suggesting a distinct response to DOX in these two different cancer types. Also, a negative correlation between PD-L1 and miR-140 was observed in DOX-treated HCT116 cells, but not in MDA-MB-231 cells.

p53 regulates katanin-p60 promoter in HCT 116 cells.

Tumor suppressor protein p53, which functions in the cell cycle, apoptosis and neuronal differentiation via transcriptional regulations of target genes or interactions with several proteins, has been associated with neurite outgrowth through microtubule re-organization. We previously demonstrated in neurons that upon p53 induction, the level of microtubule severing protein Katanin-p60 increases, indicating that p53 might be a transcriptional regulator of the KATNA1 gene encoding Katanin-p60. In this context, we firstly elucidated the activity of KATNA1 regulatory regions and endogenous KATNA1 mRNA levels in the presence or absence of p53 using HCT 116 WT and HCT 116 p53 (-/-) cells. Next, we demonstrated the binding of p53 to the KATNA1 promoter and then investigated the role of p53 on KATNA1 gene expression by ascertaining KATNA1 mRNA and Katanin-p60 protein levels upon p53 overexpression and activation in both cells. Moreover, we showed changes in microtubule network upon increased Katanin-p60 level due to p53 overexpression. Also, the changes in KATNA1 mRNA and Katanin-p60 protein levels upon p53 knockdown were investigated. Our results indicate that p53 is an activator of KATNA1 gene expression and we show that both p53 and Katanin-p60 expression have strict regulations and are maintained at balanced levels as they are vital proteins to orchestrate either survival and apoptosis or differentiation.

The fungal metabolite chaetocin is a sensitizer for pro-apoptotic therapies in glioblastoma.

Glioblastoma Multiforme (GBM) is the most common and aggressive primary brain tumor. Despite recent developments in surgery, chemo- and radio-therapy, a currently poor prognosis of GBM patients highlights an urgent need for novel treatment strategies. TRAIL (TNF Related Apoptosis Inducing Ligand) is a potent anti-cancer agent that can induce apoptosis selectively in cancer cells. GBM cells frequently develop resistance to TRAIL which renders clinical application of TRAIL therapeutics inefficient. In this study, we undertook a chemical screening approach using a library of epigenetic modifier drugs to identify compounds that could augment TRAIL response. We identified the fungal metabolite chaetocin, an inhibitor of histone methyl transferase SUV39H1, as a novel TRAIL sensitizer. Combining low subtoxic doses of chaetocin and TRAIL resulted in very potent and rapid apoptosis of GBM cells. Chaetocin also effectively sensitized GBM cells to further pro-apoptotic agents, such as FasL and BH3 mimetics. Chaetocin mediated apoptosis sensitization was achieved through ROS generation and consequent DNA damage induction that involved P53 activity. Chaetocin induced transcriptomic changes showed induction of antioxidant defense mechanisms and DNA damage response pathways. Heme Oxygenase 1 (HMOX1) was among the top upregulated genes, whose induction was ROS-dependent and HMOX1 depletion enhanced chaetocin mediated TRAIL sensitization. Finally, chaetocin and TRAIL combination treatment revealed efficacy in vivo. Taken together, our results provide a novel role for chaetocin as an apoptosis priming agent and its combination with pro-apoptotic therapies might offer new therapeutic approaches for GBMs.

Engineering antigen-specific NK cell lines against the melanoma-associated antigen tyrosinase via TCR gene transfer.

Introduction of Chimeric Antigen Receptors to NK cells has so far been the main practical method for targeting NK cells to specific surface antigens. In contrast, T cell receptor (TCR) gene delivery can supply large populations of cytotoxic T-lymphocytes (CTL) targeted against intracellular antigens. However, a major barrier in the development of safe CTL-TCR therapies exists, wherein the mispairing of endogenous and genetically transferred TCR subunits leads to formation of TCRs with off-target specificity. To overcome this and enable specific intracellular antigen targeting, we have tested the use of NK cells for TCR gene transfer to human cells. Our results show that ectopic expression of TCR α/ß chains, along with CD3 subunits, enables the functional expression of an antigen-specific TCR complex on NK cell lines NK-92 and YTS, demonstrated by using a TCR against the HLA-A2-restricted tyrosinase-derived melanoma epitope, Tyr368-377 . Most importantly, the introduction of a TCR complex to NK cell lines enables MHC-restricted, antigen-specific killing of tumor cells both in vitro and in vivo. Targeting of NK cells via TCR gene delivery stands out as a novel tool in the field of adoptive immunotherapy which can also overcome the major hurdle of "mispairing" in TCR gene therapy.

Approaches to functionally validate candidate genetic variants involved in colorectal cancer predisposition.

Most next generation sequencing (NGS) studies identified candidate genetic variants predisposing to colorectal cancer (CRC) but do not tackle its functional interpretation to unequivocally recognize a new hereditary CRC gene. Besides, germline variants in already established hereditary CRC-predisposing genes or somatic variants share the same need when trying to categorize those with relevant significance. Functional genomics approaches have an important role in identifying the causal links between genetic architecture and phenotypes, in order to decipher cellular function in health and disease. Therefore, functional interpretation of identified genetic variants by NGS platforms is now essential. Available approaches nowadays include bioinformatics, cell and molecular biology and animal models. Recent advances, such as the CRISPR-Cas9, ZFN and TALEN systems, have been already used as a powerful tool with this objective. However, the use of cell lines is of limited value due to the CRC heterogeneity and its close interaction with microenvironment. Access to tridimensional cultures or organoids and xenograft models that mimic the in vivo tissue architecture could revolutionize functional analysis. This review will focus on the application of state-of-the-art functional studies to better tackle new genes involved in germline predisposition to this neoplasm.

Nanocomposite Bioinks Based on Agarose and 2D Nanosilicates with Tunable Flow Properties and Bioactivity for 3D Bioprinting.

Three-dimensional (3D) bioprinting enables the controlled fabrication of complex constructs for tissue engineering applications and has been actively explored in recent years. However, its progress has been limited by the existing difficulties in the development of bioinks with suitable biocompatibility and mechanical properties and at the same time adaptability to the process. Herein, we describe the engineering of a nanocomposite agarose bioink with tailored properties using 2D nanosilicate additives. The suitability of agarose for 3D bioprinting has been debated due to its bioinert nature and compatibility with extrusion-based bioprinting. Nanosilicates were used to tailor the flow behavior of agarose solutions, and detailed rheological characterization of different bioink formulations was performed to elucidate the effect of nanosilicates on the flow behavior and gelation of agarose bioinks. The proper selection of nanosilicate concentration resulted in extrusion 3D printed structures with high shape fidelity and structural integrity. Moreover, the influence of addition of nanosilicates on the bioactivity of agarose was studied, and nanocomposite bioinks showed significant improvement in metabolic activity of encapsulated cells. The bioactivity of the nanocomposite bioinks was also evaluated through a cell spreading assay; the charged surfaces of nanosilicates resulted in full spreading and elongation of fibroblasts, and the extent of change in morphology of cells was found to be directly correlated with the nanosilicate concentration. Our findings suggested that engineered agarose-nanosilicate bioinks can be exploited as a new generation of hydrogel bioinks for extrusion 3D bioprinting with tunable flow properties and bioactivity.

Identification of lineage-specifying cytokines that signal all CD8+-cytotoxic-lineage-fate 'decisions' in the thymus.

T cell antigen receptor (TCR) signaling in the thymus initiates positive selection, but the CD8+-lineage fate is thought to be induced by cytokines after TCR signaling has ceased, although this remains controversial and unproven. We have identified four cytokines (IL-6, IFN-γ, TSLP and TGF-ß) that did not signal via the common γ-chain (γc) receptor but that, like IL-7 and IL-15, induced expression of the lineage-specifying transcription factor Runx3d and signaled the generation of CD8+ T cells. Elimination of in vivo signaling by all six of these 'lineage-specifying cytokines' during positive selection eliminated Runx3d expression and completely abolished the generation of CD8+ single-positive thymocytes. Thus, this study proves that signaling during positive selection by lineage-specifying cytokines is responsible for all CD8+-lineage-fate 'decisions' in the thymus.

Long noncoding RNA (lincRNA), a new paradigm in gene expression control.

Long intergenic non-coding RNAs (lincRNAs) are defined as RNA transcripts that are longer than 200 nucleotides. By definition, these RNAs must not have open reading frames that encode proteins. Many of these transcripts are encoded by RNA polymerase II, are spliced, and are poly-adenylated. This final fact indicates that there is a trove of information about lincRNAs in databases such as the Gene Expression Omnibus (GEO), which is a repository for RNAseq and microarray data. Recent experiments indicate that there are upwards of 15,000 lincRNAs encoded by the human genome. The term "intergenic" refers to the identification of these transcripts from regions of the genome that do not contain protein-encoding genes. These regions coincide with what was once labeled as the "junk DNA" portions of our genomes, which, upon careful examination by whole genome RNA sequencing experiments, clearly encode RNA transcripts. LincRNAs also contain promoter- or enhancer-associated RNAs that are gene proximal and can be either in the sense or antisense orientation, relative to the protein-coding gene with which they are associated. In this review, we describe the functions of lincRNAs playing roles in biological processes such as gene expression control, scaffold formation, and epigenetic control.

Screening of new antileukemic agents from essential oils of algae extracts and computational modeling of their interactions with intracellular signaling nodes.

Microalgae are very rich in bioactive compounds, minerals, polysaccharides, poly-unsaturated fatty acids and vitamins, and these rich constituents make microalgae an important resource for the discovery of new bioactive compounds with applications in biotechnology. In this study, we studied the antileukemic activity of several chosen microalgae species at the molecular level and assessed their potential for drug development. Here we identified Stichococcus bacillaris, Phaeodactylum tricornutum, Microcystis aeruginosa and Nannochloropsis oculata microalgae extracts with possible antileukemic agent potentials. Specifically we studied the effects of these extracts on intracellular signal nodes and apoptotic pathways. We characterized the composition of essential oils of these fifteen different algae extracts using gas chromatography-mass spectrometry (GC-MS). Finally, to identify potential molecular targets causing the phenotypic changes in leukemic cell lines, we docked a selected group of these essential oils to several key intracellular proteins. According to results of rank score algorithm, five of these essential oils analyzed might be considered as in silico plausible candidates to be used as antileukemic agents.

PATZ1 Is a DNA Damage-Responsive Transcription Factor That Inhibits p53 Function.

Insults to cellular health cause p53 protein accumulation, and loss of p53 function leads to tumorigenesis. Thus, p53 has to be tightly controlled. Here we report that the BTB/POZ domain transcription factor PATZ1 (MAZR), previously known for its transcriptional suppressor functions in T lymphocytes, is a crucial regulator of p53. The novel role of PATZ1 as an inhibitor of the p53 protein marks its gene as a proto-oncogene. PATZ1-deficient cells have reduced proliferative capacity, which we assessed by transcriptome sequencing (RNA-Seq) and real-time cell growth rate analysis. PATZ1 modifies the expression of p53 target genes associated with cell proliferation gene ontology terms. Moreover, PATZ1 regulates several genes involved in cellular adhesion and morphogenesis. Significantly, treatment with the DNA damage-inducing drug doxorubicin results in the loss of the PATZ1 transcription factor as p53 accumulates. We find that PATZ1 binds to p53 and inhibits p53-dependent transcription activation. We examine the mechanism of this functional inhibitory interaction and demonstrate that PATZ1 excludes p53 from DNA binding. This study documents PATZ1 as a novel player in the p53 pathway.