Journal of Biosciences

Tripartite motif protein 28 (TRIM28), a universal mediator of Kruppel-associated box domain zinc fingers (KRAB-ZNFs), is known to regulate DNA methylation of many repetitive elements and several imprinted loci. TRIM28 serves as a scaffold unit that is essential for the formation of stable repressor complexes. In the present study we found that TRIM28 is a binding partner for methyl-DNA binding protein Kaiso. Kaiso is a transcription factor that belongs to the BTB/POZ -zinc finger family. Recent data suggest that deficiency of Kaiso led to reduction of DNA methylation within the imprinting control region of H19/IGF2. Thus, we hypothesized that Kaiso and TRIM28 may cooperate to control methylated genes. We demonstrated that Kaiso interacts with TRIM28 via its two domains: BTB/POZ and three zinc finger domains. When bound to Kaisos zinc finger domains TRIM28 weakens their interactions with methylated DNA in vitro. Specific association of TRIM28 with BTB/POZ domain causes Kaiso hyperSUMOylation. Altogether our data describe a putative role of TRIM28 as a regulator of Kaiso activity.

Heat Shock Protein 70 (Hsp70) is a highly conserved and ubiquitous molecular chaperone that plays a central role in cellular protein machinery and stress response. Membrane-bound HSP70 has emerged as an important cancer biomarker and acts as a danger signal and elicits immune response. Hsp70 membrane expression is correlated to increased sensitivity to lysis carried out by NK cells. This study uses computational approaches to decode the interaction of Hsp70 with NK cells and determines the binding site for Hsp70 on the surface of NK cells. Our findings identified CD69 and NKP46 as the most probable binding sites for Hsp70. Additionally, we confirmed the strong binding affinity between Hsp70 and the CD94-NKG2A complex.

The ChemoReceptor-Effector Interaction Database (CREID) is a collection of bacterial chemoreceptor and effector protein and interaction data to understand the process that chemoreceptors and effectors play in various environments. Our website includes terms associated with chemosensory pathways to educate users and those involved in collaborative research to help them understand this complex biological network. It includes 2,440 proteins involved in chemoreceptor and effector systems from 7 different bacterial families with 1,996 chemoeffector interactions. It is available at https://react-creid.bicbioeng.org. Key HighlightsO_LICREID links bacterial chemoreceptors with their associated effectors. C_LIO_LIResearchers interested in what attracts or repels bacteria can use CREID as a comprehensive source for information. C_LIO_LIBiosensor developers can leverage CREID to discover better interactions for their applications. C_LIO_LICREID reveals knowledge gaps in chemoreceptor-effector interactions for both model and non-model organisms. C_LI

The RAS-RAF-MEK-ERK pathway is hyperactivated in almost all malignant melanomas, and mutations in BRAF or NRAS account for most of these cases. BRAF inhibitors are highly efficient for treating patients with BRAFV600E mutations, but tumours frequently acquire resistance within a few months. Multiple resistance mechanisms have been identified, due to mutations or network adaptations that revive ERK signalling. We have previously shown that RAF proteins inhibit the MST2 proapoptotic pathway in a kinase independent fashion. Here, we have investigated the role of the MST2 pathway in mediating resistance to BRAF inhibitors. We show that the BRAFV600E mutant protein, but not the wildtype BRAF protein, strongly binds to MST2 and inhibits MST2 pro-apoptotic signalling. Downregulation of MST2 reduces BRAF inhibitor induced apoptosis. In BRAF inhibitor resistant cell lines MST2 pathway proteins are downregulated by ubiquitination and subsequent proteasomal degradation rendering cells refractory to MST2 pathway induced apoptosis. Restoration of apoptosis can be achieved by increasing MST2 pathway protein expression using proteasome inhibitors. In summary, we show that the MST2 pathway plays a role in the acquisition of BRAF inhibitor resistance in melanoma.

Nucleolins are multifunctional proteins localized in the centrosome and nucleus and involved in the processes of microtubule nucleation. Their structure is highly conserved in organisms that are evolutionarily distant from each other. The phylogeny of the kingdom Chromista was supported by marker genes, cell morphology and cytoskeletal features. In this study we identified general patterns of the nucleolin protein structure characteristic of this group - the structure of the N-terminal and central domains, including bipartite sequences that ensure nuclear localization, as well as RNA recognition motifs. We also noted features in the primary structure of these proteins, which may affect the conformation of the protein molecule. Each of the six clades identified during phylogenetic reconstruction has structural features of the N-terminal domains, bipartite nuclear localization sequences and sequences of RNA recognition motifs. The features we described allow us to classify the studied nucleolin sequences of Chromista as nucleolin-like proteins. In this study, we described for the first time the structure of nucleolin-like proteins in Chromista.

Anastasis is a phenomenon that has been recently defined as a return from induced apoptosis. Its mechanism has not been clearly elucidated. Anastasis is thought to be involved in the development of drug resistance in cancer cells, however the distinct regulation of anastasis in normal and cancerous cells during anti-cancer therapy has not been discovered. One of the most privileged therapy strategies focuses on the drugs that are selectively cytotoxic in cancer cells but not negatively affect normal cell proliferation. This study for the first time comparatively evaluated the anastatic effect of a common synthetic cytotoxic agent, cisplatin and a natural cytotoxic agent, bee venom. The study showed that bee venom induced anastasis in normal cells (MCF10A, NIH3T3 and ARPE19) but cancer cells (MDA-MB-231 and MCF7) were irreversibly in cell death process. Liver cancer cells (HEPG2) were more resistant to bee venom-induced persistent cell death and tended to recover at higher concentrations compared to breast cancer cells. However, cisplatin induced persistent cell death in both normal and cancerous cells. Besides, selectivity indexes of bee venom in terms of IC50 values were higher than cisplatin. This study indicates that bee venom produces such an effect by selectively inducing anastasis only in normal cells suggesting that bee venom has prominent potential for cancer therapy, in particular for breast cancer, with recovery and maintenance of normal cells viability. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=136 SRC="FIGDIR/small/579605v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@4ba438org.highwire.dtl.DTLVardef@a7982org.highwire.dtl.DTLVardef@1af5950org.highwire.dtl.DTLVardef@110d199_HPS_FORMAT_FIGEXP M_FIG C_FIG

Atg1 of S. cerevisiae is a key component of autophagy encoded by ATG1 gene, involved in the process of degradation of cytosolic components through autophagy. UCC1, an F-box encoding gene is involved in the negative regulation of glyoxylate pathway via degradation of Cit2 enzyme by ubiquitin proteasome system. We investigated the genetic interaction between ATG1 and UCC1 using the gene deletion approach. The atg1{Delta}ucc1{Delta} cells showed the synthetic growth defects with abnormal budding and sensitivity to genotoxic and oxidative stress agents. Based on the observations, we report that ATG1 and UCC1 interact genetically to regulate the cell growth fitness and function in parallel pathway in cellular response to the genotoxic stress agents. The present investigation also revealed the cross talks among autophagy, ubiquitin proteasome system, and glyoxylate pathways.

The high complexity of living beings represents a difficult challenge to understand how different levels of organizations are interconnected, being especially difficult to identify how certain higher levels are affected by cellular dynamics. Important advances in ecology have shown that many phenomena can be explained through a power-law relationship, being the cellular metabolism a key component in this complexity networks scaling with a regular exponent related to body size and temperature. Here, using a novel approach we estimate the energy used to synthesize the portion of the genome that codes the metabolism searching for genomic scaling rules in five different species with higher differences in body sizes. We found that the energy of this genetic portion scales in a power-law relationship related to the mass of the species analyzed.

F-box motif encoding YDR131C is functionally uncharacterized gene which forms the complex with the SCF-E3 ligase. The F-box motif containing proteins are involved in substrate recruitment for the ubiquitination and subsequent degradation through 26S proteasome. Autophagy gene, ATG1 (ULK1in human) is a well conserved serine-threonine kinase, required for vesicle formation and cytoplasm to vacuole targeting pathway. Atg1p forms the complex with Atg13p and Atg17p during autophagy. The understanding of crosstalk between ubiquitin and autophagy pathways is crucial for synthetic lethality screen and drug targeting. Here we have conducted the study for genetic interaction between uncharacterized YDR131C and ATG1 gene representing both specific and non-specific protein degradation pathways. The single and double gene knockout strains of YDR131Cand ATG1 genes were constructed in the BY4741 genetic background and analysed for growth fitness. The strains were also evaluated for cellular growth response in presence of hydroxyurea (HU), methyl methane sulfonate (MMS), and hydrogen peroxide (H2O2) stress causing agents by spot assay. The ydr131c{Delta}atg1{Delta} showed the synthetic growth defect phenotype with floc formation in rich medium which showed floc disruption in presence of EDTA. The ydr131c{Delta}atg1{Delta} cells showed the sensitivity to stress agents HU, MMS, and H2O2 when compared with ydr131c{Delta}, atg1{Delta}, and WT cells.. Based on the observations, we report that YDR131C and ATG1 functions in parallel pathways for growth fitness and cellular growth response to stress agents. Interestingly this study also revealed the crosstalk between ubiquitination and autophagy pathways. The defects in both the pathways could lead to synthetic growth defects which may have implication for the precision medicine initiatives.

A variety of endogenous and exogenous factors induce chemical and structural alterations to cellular DNA, as well as errors occurring throughout DNA synthesis. These DNA damages are cytotoxic, miscoding, or both, and are believed to be at the origin of cancer and other age related diseases. A human cell, in addition to nuclear DNA, contains thousands copies of mitochondrial DNA (mtDNA), a double-stranded, circular molecule of 16,569 bp. It was proposed that mtDNA is a critical target for reactive oxygen species (ROS), by-products of the oxidative phosphorylation (OXPHOS), generated in the organelle during aerobic respiration. Indeed, oxidative damage to mtDNA are more extensive and persistent as compared to that of nuclear DNA. Although, transversions are the hallmarks of mutations induced by ROS, paradoxically, the majority of mtDNA mutations that occurred during ageing and cancer are transitions. Furthermore, these mutations exhibit a striking strand orientation bias: T[->]C/G[->]A transitions preferentially occur on the Light strand, whereas C[->]T/A[->]G on the Heavy strand of mtDNA. Here, we propose that the majority of mtDNA progenies, created after multiple rounds of DNA replication, are derived from the Heavy strand only, due to asymmetric replication of the DNA strand anchored to inner membrane via D-loop structure.

T cell receptor (TCR) and B cell receptor (BCR) stimulation of T and B lymphocytes, by antigen presented on an antigen-presenting cell (APC) induces the formation of the immunological synapse (IS). IS formation is associated with an initial increase in cortical filamentous actin (F-actin) at the IS, followed by a decrease in F-actin density at the central region of the IS, which contains the secretory domain. This is followed by the convergence of secretion vesicles towards the centrosome, and the polarization of the centrosome to the IS. These reversible, cortical actin cytoskeleton reorganization processes occur during lytic granule secretion in cytotoxic T lymphocytes (CTL) and natural killer (NK) cells, proteolytic granules secretion in B lymphocytes and during cytokine-containing vesicle secretion in T-helper (Th) lymphocytes. In addition, several findings obtained in T and B lymphocytes forming IS show that actin cytoskeleton reorganization also occurs at the centrosomal area. F-actin reduction at the centrosomal area appears to be associated with centrosome polarization. In this chapter we deal with the analysis of centrosomal area F-actin reorganization, as well as the centrosome polarization analysis towards the IS.

Dysregulation of cyclin-dependent kinases (CDKs) impacts cell proliferation, driving cancer. Here, we ask why the cyclin-D/CDK4 complex governs cell cycle progression through the longer G1 phase, whereas cyclin-E/CDK2 regulates the short G1/S phase transition. We consider the experimentally established high-level bursting of cyclin-E, and sustained duration of elevated cyclin-D expression in the cell, available experimental cellular and structural data, and comprehensive explicit solvent molecular dynamics simulations to provide the mechanistic foundation of the distinct activation scenarios of cyclin-D/CDK4 and cyclin-E/CDK2 in the G1 phase and G1/S transition of the cell cycle, respectively. These lead us to propose slower activation of cyclin-D/CDK4 and rapid activation of cyclin-E/CDK2. Importantly, we determine the mechanisms through which this occurs, offering innovative CDK4 drug design considerations. Our insightful mechanistic work addresses the compelling cell cycle regulation question and illuminates the distinct activation speeds in the G1 versus G1/S phases, which are crucial for cell function. StatementOur work provides an unprecedented mechanistic understanding of the distinct activation scenarios of cyclin-D/CDK4 and cyclin-E/CDK2 in cell cycle regulation, underpinning the slower activation of cyclin-D/CDK4 in the more extended G1 phase and the rapid activation of cyclin-E/CDK2 in the brief G1/S transition. Our findings address a long-standing question in cell cycle biology and suggest the design of targeted CDK4 inhibitors.

The TRPV4 cation channel, is expressed in a broad range of tissues where it participates in generation of Ca2+ signal and/or depolarization of membrane potential. Here, we identified post synaptic density protein 95 (PSD95) as an interacting protein of this epithelial Ca2+ channel using confocal microscopy analysis and immunological assay. Using co-immunoprecipitation assays, we demonstrated that PSD95 was part of the TRPV4 protein complex. PSD95 protein was specifically associated with the C-terminal tail of TRPV4 to form a complex. A TRPV4 tail deletion mutant ({Delta}DAPL871: 4d) exhibited a diminished capacity to bind PSD95. Confocal microscopy analysis suggested that apical localization of TRPV4 required PSD95-TRPV4 interaction. Our data clearly suggest that formation of a complex between TRPV4 and PSD95 can regulate TRPV4 membrane localization. Both TRPV4 Ca2+ channel and its autophagy activity of 4d were reduced by more than 80% compared to those of the TRPV4 wild type. Our observation suggests that PSD95-TRPV4 complex plays crucial roles in routing TRPV4 to the apical plasma membrane and maintaining its authentic Ca2+ channel and biological function. CapsuleO_ST_ABSBackgroundC_ST_ABSTRPV4 contain putative PDZ tail motif (DAPL871). ResultsDeletion of TRPV4 tail PDZ motif fails to interact with PSD95 PDZ III domain. ConclusionTRPV4 tail is an authentic PDZ motif to interact with PSD95. SignificanceInteraction between TRPV4 and PSD95 requires for its proper biological functions.

The symmetry and group in degeneracy of the standard genetic code (SGC) have been studied. However, the core role of equations of degree n with one unknown between symmetry and group theory has been ignored. In this study, algebraic concept was employed to abstract all genetic codons in the SGC table into equations of degree n with one unknown, and the bases in the third position of nucleotide triplets are these equations solutions. Basing upon the analysis of natural bases permutation groups and natural bases as the unit roots, some results were found that the characteristics of solvable groups imply the amount of natural bases, the relation between the first two bases in nucleotide triplets is the algebraic multiplication binary operation, and natural bases have the significance of complex number. These results gave natural bases the significance of number in the complex plane, and clarified the amount and operation in the mathematical sense, which would contribute to understand the origin and evolution of genetic code.

Physiologically, the endocannabinoids are known to reduce inflammation by decreasing production of inflammatory factors in immune and glial cells. Astrocytes secrete soluble inflammatory mediators and prolonged activation of astrocytes is associated with accelerated aging in Central Nervous System. Many reports show miRNAs as critical gene regulators in inflammation and astrogliosis in astrocytes. The aim of this study is to investigate the microRNA changes affected by Anandamide on HIV1 TAT (Trans-activator of transcription protein) stimulated normal human astrocytes. We performed global microRNA profile in TAT activated astrocytes and analysed changes on exposure to AEA. To delineate the mechanism of action we assessed with bioinformatic tools miRWalk, KEGG and Cytoscape the global microarray for significantly impacted miRNAs, and their gene targets. TAT activation in astrocytes upregulated 122 miRNAs significantly (p < 0.05). Addition of AEA in activated astrocytes downregulate the expression of 57 miRNAs significantly. Out of 122 upregulated miRNAs on TAT treatment, 37 miRNAs which were common in TAT and TAT+AEA cells showed reversal suggesting clues to critical miRNAs for the AEA-induced mitigation of neuroinflammation. Reversal in expression of selected group of miRNAs identify antagonistic pathways which are promoting anti-inflammatory environment. Pathway analysis of key 37 miRNAs show gene targets that regulate inflammation and senescence.

Chromosome transmission fidelity factor, Ctf4 in S. cerevisiae associates with replication fork and helps in the sister chromatid cohesion. At the replication fork, Ctf4 links DNA helicase with the DNA polymerase. The absence of Ctf4 invokes replication checkpoint in the cells. The Saf1 of S.cerevisiae interacts with Skp1 of SCF-E3 ligase though F box-motif and ubiquitinates the adenine deaminase Aah1 during phase transition due to nutrient stress. The genetic interaction between the CTF4 and SAF1 has not been studied. Here we report genetic interaction between CTF4 and SAF1 which impacts the growth fitness and response to stress. The single and double gene deletions of SAF1 and CTF4 were constructed in the BY4741 genetic background. The strains were tested for growth on rich media and media containing stress causing agents. The saf1{Delta}ctf4{Delta} cells with reduced cell size showed the fastest growth phenotype on YPD medium when compared with the saf1{Delta}, ctf4{Delta}, and WT. The saf1{Delta}ctf4{Delta} cells also showed the tolerance to MMS, NaCl, Glycerol, SDS, Calcofluor white, H2O2, DMSO, Benomyl, and Nocodazole when compared with the saf1{Delta}, ctf4{Delta}, and WT cells. However, saf1{Delta}ctf4{Delta} cells showed the sensitivity to HU when compared with WT and saf1{Delta}. Based on these observations we suggest that SAF1 and CTF4 interact genetically to regulate the cell size, growth and stress response.

BackgroundTerpenoids form a large pool of highly diverse organic compounds possessing several economically important properties, including nutritional, aromatic, and pharmacological properties. The DXP pathways end enzyme, nuclear distribution protein (NudF), interacting with isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), is critical for the synthesis of isoprenol/prenol/downstream compounds. The enzyme is yet to be thoroughly investigated to increase the overall yield of terpenoids in the Bacillus subtilis, which is widely used in industry and is generally regarded as safe (GRAS) bacterium. The study aims to analyze the evolutionary conservation across the active site, and map the key residues for mutagenesis studies. The study would allow us customize the metabolic load towards the synthesis of prenol or isoprenol or any of the downstream molecules. ResultsThe 37-sequence dataset, extracted from 103 Bacillus subtilis entries, show a high phylogenetic divergence, and only six one-motif sequences ASB92783.1, ASB69297.1, ASB56714.1, AOR97677.1, AOL97023.1, and OAZ71765.1 show monophyly relationship, unlike a complete polyphyly relationship between the other 31 three-motif sequences. Further, only 47 of 179 residues of the representative sequence CUB50584.1 are observed to be significantly conserved. Docking analysis shows a preferential bias of ADP-ribose pyrophosphatase towards IPP, and a nearly 3-fold energetic difference is observed between IPP and DMAPP. Computational saturation mutagenesis of the seven hotspot residues identifies two key positions LYS78 and PHE116, encoded within loop1 and loop7, majorly interact with the ligands DMAPP and IPP, and their mutants K78I/K78L and PHE116D/PHE116E are found to stabilize the overall conformation. The loops are hereby shown to play a regulatory role in guiding the promiscuity of NudF towards a specific ligand. ConclusionThe study map the phylogenetic relationship between the 37 representative B.subtiis NudF sequences, and through sequence conservation, structural contact map, topological flexibility, and saturation mutagenesis of the active site residues, the essential residues regulating the interaction of NudF with IPP/DMAPP are deciphered. The study robustly screens its mutational landscape and localizes the two crucial residues LYS78 and PHE116 for directing the mutagenesis studies. The preliminary docking and simulation results also suggest a preferential bias of ADP-ribose pyrophosphatase towards IPP over DMAPP. The findings would pave the way for the development of novel enzyme variants with highly improved catalytic ability for the large-scale bioproduction of specific terpenoids with significant neutraceutical or commercial value.

Transposable elements (TEs) are abundantly present in eukaryotic genomes and can be likened to parasites colonizing a genome due to their properties. From this perspective, a population-based approach has been developed to model interactions between TEs and a genome population. The distribution of TEs within a population of genomes is studied over the long term to understand the mechanisms allowing TEs to persist despite their deleterious effects on genomes. Under this single restrictive assumption, the results show that the population of TEs can persist for a very long time within the genome population, when the genome population is highly diverse in terms of the distribution of TEs quantities. When there is no mechanism for silencing TEs, the proposed model of asexual reproduction either purges TEs or leads to co-extinction of genomes and TEs. On the other hand, with a high proportion of silenced TEs, the population of TEs can be maintained for a long time in the population of genomes.

Base substitution mutations such as transition (ti) and transversion (tv) in organisms are major driving force in molecular evolution. In this study, different possible types of base pairing that can cause ti and tv were investigated using the density functional theory (DFT) method. The chemical structures of bases as well as base pairs were optimized using B3LYP hybrid functional along with 6-31G(d,p) basis set. We performed single point energy calculation of all optimized species using the same functional but combined with higher diffuse and polarized basis set i.e. 6-311++G(d,p) to get more refined energy of all species. The binding energy of various base pairs was calculated considering basis set superposition error (BSSE) as well as without BSSE. The binding energy of the base pairs leading to ti was found to be more stable than that of the base pairs leading to tv. This was interesting considering the observations in organisms that tis are more frequent than tvs. Among the base pairs leading to the same ti, G(keto): T (enol) base pair was found to be more stable than A(imino):C(amino) base pair. This theoretical study of binding energy of different base pairs using the DFT method has provided additional evidences in support to the biological observations of a higher transition rate than transversion in genomes.

Replication of DNA is an essential process in all domains of life. A protein often involved without exception in replication is the sliding clamp. The sliding clamp encircles the DNA and helps replicative polymerase stay attached to the replication machinery increasing the processivity of the polymerase. In eukaryotes and archaea the sliding clamp is called the Proliferating Cell Nuclear Antigen (PCNA) and consists of two domains. This PCNA forms a trimer encircling the DNA as a hexamer. In bacteria, the structure of the sliding clamp is highly conserved, but the protein itself, called beta clamp, contains three domains, which dimerize to form a hexamer. The bulk of literature touts a conservation of the structure of the sliding clamp, but fails to recognize conservation of protein sequence among sliding clamps. In this paper we have used PSI blast to the second interation in NCBI to show a statistically significant sequence homology between Pyrococcus furiosus PCNA and Kallipyga gabonensis beta clamp. The last two domains of beta clamp align with the two domains of PCNA. This homology data demonstrates that PCNA and beta clamp arose from a common ancestor. In this paper, we have further used beta clamp and PCNA sequences from diverse bacteria, archaea and eukarya to build maximum likelihood phylogenetic tree. Most, but not all, species in different domains of life harbor one sliding clamp from vertical inheritance. Some of these species that have two or more sliding clamps have acquired them from gene duplication or horizontal gene transfer events.