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Supplementary Figure 4 from Repression of RUNX1 Activity by EVI1: A New Role of EVI1 in Leukemogenesis

Supplementary Figure 5 from Repression of RUNX1 Activity by EVI1: A New Role of EVI1 in Leukemogenesis

Supplementary Figure 2 from Repression of RUNX1 Activity by EVI1: A New Role of EVI1 in Leukemogenesis

Supplementary Figure 3 from Repression of RUNX1 Activity by EVI1: A New Role of EVI1 in Leukemogenesis

Supplementary Figure 1 from Repression of RUNX1 Activity by EVI1: A New Role of EVI1 in Leukemogenesis

Recurring chromosomal translocations observed in human leukemia often result in the expression of fusion proteins that are DNA-binding transcription factors. These altered proteins acquire new dimerization properties that result in the assembly of inappropriate multimeric transcription complexes that deregulate hematopoietic programs and induce leukemogenesis. Recently, we reported that the fusion protein AML1/MDS1/EVI1 (AME), a product of a t(3;21)(q26;q22) associated with chronic myelogenous leukemia and acute myelogenous leukemia, displays a complex pattern of self-interaction. Here, we show that the 8th zinc finger motif of MDS1/EVI1 is an oligomerization domain involved not only in interaction of AME with itself but also in interactions with the parental proteins, RUNX1 and MDS1/EVI1, from which AME is generated. Because the 8th zinc finger motif is also present in the oncoprotein EVI1, we have evaluated the effects of the interaction between RUNX1 and EVI1 in vitro and in vivo. We found that in vitro, this interaction alters the ability of RUNX1 to bind to DNA and to regulate a reporter gene, whereas in vivo, the expression of the isolated 8th zinc finger motif of EVI1 is sufficient to block the granulocyte colony-stimulating factor–induced differentiation of 32Dcl3 cells, leading to cell death. As EVI1 is not detected in normal bone marrow cells, these data suggest that its inappropriate expression could contribute to hematopoietic transformation in part by a new mechanism that involves EVI1 association with key hematopoietic regulators, leading to their functional impairment. [Cancer Res 2007;67(12):5658–66]

We have developed a new series of simple biaryl piperidine derivatives (11-19) based on biaryl naphthylisoquinoline alkaloid Ealamine-A. The target compounds were synthesized, analyzed by spectral data, and evaluated for antileishmanial activity against Leishmania donovani strain Ag83 by MTT assay. The compounds have shown the best to moderate antileishmanial activity. The 5'-fluoro-2'-methoxyphenyl derivative 14 and 3',5'-difluorophenyl derivative 16 have inhibited the promastigotes by 86 % and 85 % after 24 h and 92 % and 91 % after 48 h incubation, respectively, at 400 μM concentration. The % inhibition was lower with the lowering of the concentration and increased with the incubation time. Compounds 12, 15, and 18 have solubility issues and proved to be less active than the rest of the compounds. Molecular docking studies were performed on selective active compounds and the results indicate that these compounds may act by binding to the Leishmanolysin and the docking scores are in good correlation with the antileishmanial activity. These results provide an initial insight into the design of new therapeutics for neglected tropical diseases.

The visceral leishmaniasis is caused by L. donovani, a neglected tropical disease with an estimated number of 500,000 cases worldwide. Apart from the absence of effective vaccine, the available drugs have limitations like toxic side effects and emergence of drug resistance. The genome of Leishmania is remarkably challenged by the oxidative stress present inside the human macrophage. To maintain genomic integrity, a number of specialized DNA repair pathways assist in the recognition and repair of damaged DNA. In general, Base Excision Repair (BER) plays an essential role in the maintenance of genomic stability. We demonstrate here that the treatment of L. donovani with oxidative agents causes DNA damage and upregulation of Polβ. On the other hand, parasite overexpressing Polβ shows more resistance against Amp B, H2O2 and menadione as compared to wild type cells. We also observed a higher infectivity in the parasites that overexpress Polβ. The upregulation of Polβ was also found in stationary phase and axenic amastigote of L. donovani. Overall, we propose that Polβ is crucial for infectivity and survival of the parasite. Discovery of specific inhibitors against Polβ could offer an attractive strategy against leishmaniasis.

5,6-Dihydroxy-5,6-dihydrothymine (thymine glycol) and 7,8-dihydro-8-oxo-20-deoxyguanosine (8-oxodG) are major DNA damage lesions produced by endogenous oxidative stress, as well as inflicted by carcinogens and ionizing radiation. The processing of Tg:G mismatch and 8-oxodG in close proximity of each other in a bistranded clustered environment in DNA oligomer duplexes as well as in a nucleosome core particle (NCP) model are reported here. The processing of the lesions was evaluated by purified enzyme cocktails of hNTH1 and hOGG1 as well as with a HeLa cell extract. Interestingly, the yield of double-strand breaks (DSBs) resulting from the processing of the bistranded lesions are appreciably lower when the DNA is treated with the HeLa cell extract compared with the relevant purified enzyme cocktail in both models. Clustered bistranded lesions become more repair refractive when reconstituted as an NCP. This indicates a complex interplay between the repair enzymes that influence the processing of the bistranded cluster damage positively to avoid the formation of DSBs under cellular conditions. In addition to position and orientation of the lesions, the type of the lesions in the cluster environment in DNA along with the relative abundance of the lesion-specific enzymes in the cells strongly prevents the processing of the oxidized nucleobases.

The occurrence of 7,8-dihydro-8-oxo-2'deoxyguanosine (8-oxodG), thymine glycol:guanine (Tg:G) mismatch and abasic site DNA damage lesions in close proximity induce repair refractive multicomponent clustered DNA damage. Herein, the influence of abasic sites in the processing of 8-oxodG lesion and Tg:G mismatch bistranded cluster is evaluated. Abasic sites are found to impart conformational destabilization that appreciably hinders the repair activity of the other lesions whenever present in a cluster combination. The repair process reduces the formation of double strand breaks (DSBs) and renders this three-lesion combination a non-DSB forming cluster. The stability of the DNA duplex harbouring these three lesions is highly compromised due to altered base helicity and base stacking phenomena leading to impaired repair.

DNA derived well-controlled arrangement of porphyrins has emerged as promising hybrid nanostructures. Exceptional biocompatibility and DNA-directed surface addressability coupled with rich symmetry features of the porphyrin have made these hybrid nanostructures attractive candidates for potential biomedical and biotechnological applications. However, the noteworthy photophysical properties of porphyrin and related molecules when present in DNA based nanostructures are yet to be explored fully and should be a matter of intense research that may unearth a plethora of interesting applications of these nanostructures. Herein, we demonstrate the construction of novel self-assembled DNA-porphyrin hybrid nanonetworks that utilize the porphyrin core for antibacterial applications. Porphyrin derivative with four pendant NH2 groups was synthesized and conjugated with the 5′-PO4 of ss-DNA by solution phase phosphoramidation coupling reaction. The conjugation was followed by DNA hybridization mediated self-assembly to form DNA-porphyrin hybrid nanonetwork. The enhanced antimicrobial property of the nanonetwork was envisioned following light irradiation at relevant wavelength. In line with this, comparative antimicrobial activities against gram-negative (Escherichia coli BL-21) and gram-positive bacteria (Staphylococcus aureus) have been studied. Interestingly, DNA-porphyrin nanonetwork afforded highly efficient and coherent photoinduced reactive oxygen species (ROS) generation to display antimicrobial activity against Staphylococcus aureus. The escalated and coherent ROS generation from the nanonetworks was attributed to the ordered placement of the porphyrins that inhibits self-quenching. Our work points out to a good alternative for antibiotic free strategies for preservation of biological materials and other applications.

The construction of DNA-carbon dot (CD) hybrid hydrogel for targeted and sustained release of drug molecules is reported here. Amine functionalized carbon dots were conjugated to 5′-phosphate termini of Cytosine (C) rich ssDNA by phosphoramidate linkage. As a prototype, chemotherapeutic drug Doxorubicin (Dox) was loaded and enclosed in hydrogel that acts as a container for sustained release of the drug. Apart from acting as a cross linker for network formation, CDs also participate in encapsulating the drug by electrostatic interaction along with DNA. Moreover, photophysical properties of CD potentially enable tracking of hydrogel dissolution and drug cargo loading in hydrogel. The visually detectable sol-gel transition of CD-DNA hybrid hydrogel was achieved by varying the pH of the solution from alkaline to neutral. The in vitro time and pH dependent release profile of the drug from hydrogel was studied. While hydrogel was found to be stable for a month at normal physiological pH, complete dissolution and sustained release of the drug molecules were achieved over 10–11 days in acidic pH, relevant to tumor microenvironment. The cell viability assay performed on HeLa cells shows their effective slow killing in presence of Dox loaded hydrogel owing to favorable acidic pH for hydrogel disruption.

Non-targeted photosensitizers lack selectivity that undermines the potential use of photodynamic therapy (PDT). Herein, we report the DNA mediated assembly of a ZnSe/ZnS quantum dot (QD)-photosensitizer (PS)-Mucin 1(MUC1) aptamer conjugate for targeting the MUC1 cancer biomarker and simultaneous generation of reactive oxygen species (ROS). A photosensitizer, protoporphyrin IX (PpIX), was conjugated to a single stranded DNA and self-assembled to a complementary strand that was conjugated to a QD and harboring a MUC1 aptamer sequence. A multistep fluorescence resonance energy transfer (FRET) is shown that involves the QD, PpIX and covalently linked CF™ 633 amine dye (CF dye) to the MUC1 peptide that tracks the potency of the aptamer to attach itself with the MUC1 peptide. Since the absorption spectra of the CF dye overlap with the emission spectra of PpIX, the former acts as an acceptor to PpIX forming a second FRET pair when the dye labeled MUC1 binds to the aptamer. The binding of the QD-PpIX nanoassemblies with MUC1 through the aptamer was further confirmed by gel electrophoresis and circular dichroism studies. The selective photodamage of MUC1 expressing HeLa cervical cancer cells through ROS generation in the presence of the QD-PpIX FRET probe upon irradiation is successfully demonstrated.

Leishmania donovaniis an intracellular protozoan parasite that causes endemic tropical disease visceral leishmaniasis (VL). Present drugs used against this fatal disease are facing resistance and toxicity issues. Survival of leishmania inside the host cells depends on the parasite’s capacity to cope up with highly oxidative environment. Base excision repair (BER) pathway inL. donovaniremains unexplored. We studied uracil DNA glycosylase (UNG), the key enzyme involved in BER pathway, and found that the glycosylase activity of recombinant LdUNG (Leishmania donovaniUNG) expressed inE. coliis in sync with the activity of the parasite lysate under different reaction conditions. Overexpression of UNG in the parasite enhances its tolerance towards various agents which produce reactive oxygen species (ROS) and shows a higher infectivity in macrophages. Surprisingly, exposure of parasite to amphotericin B and sodium antimony gluconate upregulates the expression of UNG. Further, we found that the drug resistant parasites isolated from VL patients show higher expression of UNG. Mechanisms of action of some currently used drugs include accumulation of ROS. Our findings strongly suggest that targeting LdUNG would be an attractive therapeutic strategy as well as potential measure to tackle the problem of drug resistance in the treatment of leishmaniasis.

Leishmania donovani is an intracellular protozoan parasite that causes endemic tropical disease visceral leishmaniasis (VL). Present drugs used against this fatal disease are facing resistance and toxicity issues. Survival of leishmania inside the host cells depends on the parasite's capacity to cope up with highly oxidative environment. Base excision repair (BER) pathway in L. donovani remains unexplored. We studied uracil DNA glycosylase (UNG), the key enzyme involved in BER pathway, and found that the glycosylase activity of recombinant LdUNG (Leishmania donovani UNG) expressed in E. coli is in sync with the activity of the parasite lysate under different reaction conditions. Overexpression of UNG in the parasite enhances its tolerance towards various agents which produce reactive oxygen species (ROS) and shows a higher infectivity in macrophages. Surprisingly, exposure of parasite to amphotericin B and sodium antimony gluconate upregulates the expression of UNG. Further, we found that the drug resistant parasites isolated from VL patients show higher expression of UNG. Mechanisms of action of some currently used drugs include accumulation of ROS. Our findings strongly suggest that targeting LdUNG would be an attractive therapeutic strategy as well as potential measure to tackle the problem of drug resistance in the treatment of leishmaniasis.

Currently, there is no approved vaccine for visceral leishmaniasis (VL) caused by L. donovani. The ability to manipulate Leishmania genome by eliminating or introducing genes necessary for parasites’ survival considered as the powerful strategy to generate the live attenuated vaccine. In the present study fructose-1,6-bisphosphatase (LdFBPase) gene deleted L. donovani (Δfbpase) was generated using homologous gene replacement strategy. Though LdFBPase gene deletion (Δfbpase) does not affect the growth of parasite in the promastigote form but axenic amastigotes display a marked reduction in their capacity to multiply in vitro inside macrophages and in vivo in Balb/c mice. Though Δfbpase L. donovani parasite persisted in BALB/c mice up to 12 weeks but was unable to cause infection, we tested its ability to protect against a virulent L. donovani challenge. Notably, intraperitoneal immunisation with live Δfbpase parasites displayed the reduction of parasites load in mice spleen and liver post challenge. Moreover, immunised BALB/c mice showed a reversal of T cell anergy and high levels of NO production that result in the killing of the parasite. A significant, correlation was found between parasite clearance and elevated IFNγ, IL12, and IFNγ/IL10 ratio compared to IL10 and TGFβ in immunised and challenged mice. Results suggested the generation of protective Th1 type immune response which induced significant parasite clearance at 12-week, as well as 16 weeks post, challenged immunised mice, signifying sustained immunity. Therefore, we propose that Δfbpase L. donovani parasites can be a live attenuated vaccine candidate for VL and a good model to understand the correlatives of protection in visceral leishmaniasis.

Histone post-translational modifications (PTMs) such as acetylation and methylation are known to affect chromatin higher order structures. Primary targets of these modifications include basic residues present at N-terminus tail region of core histones. Four histone acetyltransferase (HAT) genes have been identified in trypanosomatids. HAT1, HAT3 and HAT4 of Leishmania donovani have been partially characterized. However, there is no report about HAT2 of Leishmania donovani. Lysine residues present on the N-terminal tail of Leishmania donovani histone H4 are conserved in other trypanosomatids and humans. PTMs of lysines modulate various functions at chromatin level. The four histone acetyltransferases encoded in Leishmania genome were over-expressed to analyse their functional activity. All four HATs were found actively acetylating core histones H3/H4. Similar to L. donovani HAT3 and HAT4, HAT2 was found to be a member of MYST family protein and have SAS2 type domain. Over-expression of HAT2 significantly increases acetylation of H4K4. To analyse the effect of HAT2 over-expression on chromatin accessibility, micrococcal nuclease digestion assay was performed. MNase digestion resulted in a higher proportion of the mononucleosomes and dinucleosomes in HAT2 over-expressing cells as compared to WT L. donovani cells. Acetylation of lysine-4 neutralizes the amino terminal region of histone H4. This weakens its interaction with neighbouring nucleosomes and the linker DNA. HAT2 over-expression in L. donovani resulted in highly accessible chromatin suggesting chromatin decondensation. HAT2 may have an important role to play in global regulation of transcription in L. donovani. Better understanding of these epigenetic determinants of parasite would help in designing novel therapeutic strategies.

Plasma gelsolin (pGSN) is a multifunctional protein involved mainly in severing and clearing of actin filaments. Its level correlates with inflammation and several diseases making it a potential biomarker of diagnostic and prognostic values. The pGSN level in groups of treated and untreated HIV-1-infected Indian patients is investigated in this study. This study aims at investigating the levels of pGSN in HIV-1-infected patients across different age, sex, severity of disease, and treatment status. Blood samples of 213 patients were analyzed for CD4 counts by flow cytometry and pGSN was quantified by enzyme-linked immunosorbent assay (ELISA). The level of pGSN is significantly increased in HIV-1 infected patients (227.2 ± 54.3 μg/ml) compared to healthy volunteers (167.9 ± 61.8 μg/ml). The level correlates with CD4 cell counts as patients with lower CD4 counts showed higher pGSN levels and vice versa. Gender does not affect pGSN level; however, antiretroviral (ARV) treatment reduces pGSN toward normal. Within low CD4 cell count group, the untreated patients have 52% higher pGSN than healthy volunteers, whereas with treatment, the difference reduces to 24%. Similarly, high CD4 cell count (350 cells/mm

Various physiological stimuli trigger the conversion of noninfective Leishmania donovani promastigotes to the infective form. Here, we present the first evidence of the effect of glucose starvation, on virulence and survival of these parasites. Glucose starvation resulted in a decrease in metabolically active parasites and their proliferation. However, this was reversed by supplementation of gluconeogenic amino acids. Glucose starvation induced metacyclogenesis and enhanced virulence through protein kinase A regulatory subunit (LdPKAR1) mediated autophagy. Glucose starvation driven oxidative stress upregulated the antioxidant machinery, culminating in increased infectivity and greater parasitic load in primary macrophages. Interestingly, phosphoenolpyruvate carboxykinase (LdPEPCK), a gluconeogenic enzyme, exhibited the highest activity under glucose starvation to regulate growth of L. donovani by alternatively utilising amino acids. Deletion of LdPEPCK (Δpepck) decreased virulent traits and parasitic load in primary macrophages but increased autophagosome formation in the mutant parasites. Furthermore, Δpepck parasites failed to activate the Pentose Phosphate Pathway shunt, abrogating NADPH/NADP+ homoeostasis, conferring increased susceptibility towards oxidants following glucose starvation. In conclusion, this study showed that L. donovani undertakes metabolic rearrangements via gluconeogenesis under glucose starvation for acquiring virulence and its survival in the hostile environment.

Recurring chromosomal translocations observed in human leukemia often result in the expression of fusion proteins that are DNA-binding transcription factors. These altered proteins acquire new dimerization properties that result in the assembly of inappropriate multimeric transcription complexes that deregulate hematopoietic programs and induce leukemogenesis. Recently, we reported that the fusion protein AML1/MDS1/EVI1 (AME), a product of a t(3;21)(q26;q22) associated with chronic myelogenous leukemia and acute myelogenous leukemia, displays a complex pattern of self-interaction. Here, we show that the 8th zinc finger motif of MDS1/EVI1 is an oligomerization domain involved not only in interaction of AME with itself but also in interactions with the parental proteins, RUNX1 and MDS1/EVI1, from which AME is generated. Because the 8th zinc finger motif is also present in the oncoprotein EVI1, we have evaluated the effects of the interaction between RUNX1 and EVI1 in vitro and in vivo. We found that in vitro, this interaction alters the ability of RUNX1 to bind to DNA and to regulate a reporter gene, whereas in vivo, the expression of the isolated 8th zinc finger motif of EVI1 is sufficient to block the granulocyte colony-stimulating factor–induced differentiation of 32Dcl3 cells, leading to cell death. As EVI1 is not detected in normal bone marrow cells, these data suggest that its inappropriate expression could contribute to hematopoietic transformation in part by a new mechanism that involves EVI1 association with key hematopoietic regulators, leading to their functional impairment. [Cancer Res 2007;67(12):5658–66]

Adenosine, an endogenous purine nucleoside is one such extracellular signalling molecule whose role in regulation of anti-inflammatory cytokines and immune pathogenicity in visceral leishmaniasis is not fully understood. Here, we investigated the relationship between Leishmania donovani infection and expression of A2B receptor on monocytes in VL patients in their pre and post treatment stage. We also investigated the molecular mechanisms influencing the interaction between immunopathogenicity and infection by exposing Leishmania donovani pulsed macrophages to Adenosine. A direct correlation of up-regulated A2B expression on monocytes with increased parasite load was also observed. Our results also suggested that A2B receptor activation is critically required for the stimulatory effect of adenosine on IL-10 production and suppression of nitric oxide release. The stimulatory effect of adenosine on Leishmania donovani induced IL-10 production required ERK1/2 activation and is p-38 MAPK independent.

L-Asparaginases belong to a family of amidohydrolases that catalyze the conversion of L-asparagine into L-aspartic acid and ammonia. Although bacterial L-asparaginases have been used extensively as anti-leukemic agents, their possible role as potential drug targets for pathogenic organisms has not been explored. The presence of genes coding for putative L-asparaginase enzymes in the Leishmania donovani genome hinted towards the specific role of these enzymes in extending survival benefit to the organism. To investigate whether this enzyme can serve as a potential drug target against the Leishmania pathogen, we obtained structural models of one of the putative Leishmania L-asparaginase I (LdAI). Using an integrated computational approach involving molecular modelling, docking and molecular dynamics simulations, we found crucial differences between catalytic residues of LdAI as compared to bacterial L-asparaginases. The deviation from the canonical acid–base pair at triad I, along with the structural reorganization of a β-hairpin loop in the presence of a substrate, indicated an altogether new mechanism of action of the LdAI enzyme. Moreover, the finding of compositional and functional differences between LdAI and human asparaginase was used as a criterion to identify specific small molecule inhibitors. Through virtual screening of a library of 11 438 compounds, we report five compounds that showed favorable interactions with the active pocket of LdAI, without adversely affecting human asparaginase. One of these compounds when tested on cultured Leishmania promastigotes displayed a promising leishmanicidal effect. Overall, our work not only provides first hand mechanistic insights of LdAI but also proposes five strongly active compounds which may prove as effective anti-leishmaniasis molecules.

Converging studies from many investigators indicate that RUNX1 has a critical role in the correct maintenance of essential cellular functions during embryonic development and after birth. The discovery that this gene is also frequently mutated in human leukemia has increased the interest in the role that RUNX1 plays in both normal and transforming pathways. Here, we provide an overview of the many roles of RUNX1 in hematopoietic self-renewal and differentiation and summarize the information that is currently available on the many mechanisms of RUNX1 deregulation in human leukemia.

Acute myeloid leukemia 1 (AML1) belongs to a family of DNA-binding proteins highly conserved through evolution. AML1 regulates the expression of several hematopoietic genes and is essential for murine fetal liver hematopoiesis. We report here that the histone methyltransferase SUV39H1, a mammalian ortholog of the Drosophila melanogaster SU(VAR) 3-9, forms complex with AML1. SUV39H1 methylates lysine 9 of the histone protein H3 leading to the formation of the high-affinity binding site on chromatin for proteins of the heterochromatin protein 1 family (HP1). The interaction of AML1 with SUV39H1 requires the N-terminus of AML1 where the Runt domain is located. Binding of AML1 to SUV39H1 abrogates the transactivating and DNA-binding properties of AML1 and dissociates the net-like nuclear structure of AML1. It has been reported that AML1 is capable of interaction with histone acetyl transferases (CBP, p300, and MOZ) and with component of the histone deacetylase complex (Sin3), and that the interaction with these coregulators affects the strength of AML1 in promoter regulation. Our data suggest that other enzymes are also involved in gene regulation by AML1 activity by modulating the affinity of AML1 for DNA.

Molecular mechanisms of multidrug resistance in Vibrio cholerae belonging to non-O1, non-O139 serogroups isolated during 1997 to 1998 in Calcutta, India, were investigated. Out of the 94 strains examined, 22 strains were found to have class I integrons. The gene cassettes identified were dfrA1 , dfrA15 , dfrA5 , and dfrA12 for trimethoprim; aac ( 6 ′) -Ib for amikacin and tobramycin; aadA1 and aadA2 for streptomycin and spectinomycin; and ereA2 for erythromycin resistance. To our knowledge, this is the first report of the presence of dfrA5 , dfrA12 , aac ( 6 ′) -Ib, and ereA2 cassettes in class I integrons of V. cholerae . Forty-three of 94 strains also had plasmids, and out of these, 14 contained both class I integrons and plasmids. Pulsed-field gel electrophoresis followed by Southern hybridization revealed that in the 14 plasmid-bearing strains, class I integrons resided either on chromosomes, on plasmids, or on both. Our results indicated that besides class I integrons and plasmids, a conjugative transposon element, SXT, possibly contributed to the multiple antibiotic resistance.

Leishmania donovani encounters oxidative environment in the host macrophage and expected to have robust DNA repair mechanisms. Base Excision Repair (BER), a predominant repair pathway in L. donovani remains unexplored. Presence of mitochondria in eukaryotes has been projected as a symbiotic relationship since long and the role of DNA polymerase β in repair of mitochondrial DNA has gained importance in recent past. We ectopically expressed Leishmania DNA polymerase β (LdPolβ) under inducible promoter in E. coli and found it is biologically active in vitro by using pUC19 as substrate. Further we checked its effect on sensitivity of E. coli to UV rays. We find that heterologous LdPolβ slows down the growth of E. coli and surprisingly, could protect it from lethal effects of UV to a large extent. Co-expression of leishmania DNA Ligase IIIα (LdLigIIIα) has a synergistic effect on survival advantage offered by LdPolβ. Survival advantage given LdPolβ in E. coli is reconfirmed by FACS analysis. Our observations indicate that LdPolβ is crucial for handling ROS induced toxicity inside the mitochondria of the parasite and for its survival inside host macrophage. This studied may lead to explore for finding of the importance of LdPolβ in survival against DNA damaging agents in L. donovani and its role in pathogenesis of leishmaniasis, it would help to discover new target and development of newer drug against Leishmaniasis.

Phagocytic cells produce reactive oxygen and nitrogen species (ROS & RNS) as the most common arsenal to kill intracellular pathogens. Leishmania, an obligate intracellular pathogen also confronts this antimicrobial assault during the early phase of infection but nevertheless is able to survive these attacks and proliferate in macrophage. Adaptation of Leishmania to the toxic effects of ROS and RNS, involves a rapid change in the parasite proteome to combat the host defense response that macrophage mount in combating pathogen. To understand the events associated with combating ROS and RNS species, we performed a proteomic analysis of L. donovani promastigotes treated with sub-lethal doses of menadione (ROS), S-nitroso-N-acetylpenicillamine (RNS) or combination of both compounds. Proteomic changes triggered by these reagents were evaluated by iTRAQ labeling and subsequent LC-MALDI-TOF/TOF-MS analysis. Across the 3 stress conditions, the quantitative analysis identified changes in the proteins which encompass ~20% of the parasite proteome. Major changes were observed in enzymatic machinery of pathways involved in maintaining redox homeostasis, trypanothione metabolism, oxidative phosphorylation, superoxide metabolism, mitochondrial respiration process and other essential metabolic pathways. These observations shed light on how Leishmania promastigotes counter ROS and RNS effects during the initial stage of infection. This article is part of a Special Issue entitled: From protein structures to clinical applications.

RUNX1 (AML1, CBFalpha2, PEBP2alphaB) is a transcription factor essential for the establishment of the hematopoietic stem cell. It is generally thought that RUNX1 exists as a monomer that regulates hematopoietic differentiation by interacting with tissue-specific factors and its DNA consensus through its N terminus. RUNX1 is frequently altered in human leukemia by gene fusions or point mutations. In general, these alterations do not affect the N terminus of the protein, and it is unclear how they consistently lead to hematopoietic transformation and leukemia. Here we report that RUNX1 homodimerizes through a mechanism involving C terminus-C terminus interaction. This RUNX1-RUNX1 interaction regulates the activity of the protein in reporter gene assays and modulates its ability to induce hematopoietic differentiation of hematopoietic cell lines. The promoters of genes regulated by RUNX1 often contain multiple RUNX1 binding sites. This arrangement suggests that RUNX1 could homodimerize to bring and hold together distant chromatin sites and factors and that if the dimerization region is removed by gene fusions or is altered by point mutations, as observed in leukemia, the ability of RUNX1 to regulate differentiation could be impaired.

Transcription repression in eukaryotes is mediated by a wide variety of transcription factors that usually recruit corepressors and form corepressor complexes at the specific promoter sites. One of these corepressors is the C-terminal-binding protein (CtBP) which was first identified as a protein that binds to the C-terminal region of the adenovirus E1A protein. CtBP has a strong role in both development and oncogenesis. Till date, the mechanism of transcription repression by CtBP is unknown. Here, we report that CtBP physically interacts in vivo with HAT enzymes from different families. The vast majority of the HAT enzymes have a potential consensus site for CtBP binding within the bromodomain but we show that additional site(s) exists for CBP. The interaction between CtBP and CBP is functionally important and leads to impairment of histone H3 acetylation by CBP at specific lysine residues (Lys9, Lys14, and Lys18) in a dose-dependent and NADH-dependent manner. Based on these results, we propose that CtBP1 mediates repression by blocking histone acetylation by HAT complexes.

Lysine acetyltransferases modulate the activity of many genes by modifying the lysine residues of both core histones and transcription-related factors. These modifications are tightly controlled in the cell because they are involved in vital processes such as cell cycle progression, differentiation, and apoptosis. Therefore, any deregulation of acetylation/deacetylation equilibrium or inappropriate modifications could lead to different diseases. Since previous studies have shown that some oncoproteins also undergo this modification, acetylation could be involved in the processes of cell transformation and oncogenesis. Here, we report that AML1/MDS1/EVI1 (AME), a repressor produced by the t(3;21) associated with human leukemia, physically interacts with the acetyltransferases P/CAF and GCN5. Our data suggest that AME has at least two binding sites for these acetyltransferases, one of which is in the Runt domain. Both P/CAF and GCN5 efficiently acetylate AME in vivo in the central region. AME acetylation has no effect on its interaction with the co-repressor CtBP1. Finally, we demonstrate that the co-expression of AME and either P/CAF or GCN5 abrogates the repression of an AML1-dependent reporter gene.

Myelodysplastic syndrome (MDS) comprises a group of disorders with overlapping features. The majority of recurring chromosomal abnormalities associated with MDS are large deletions. There are two genes, RUNX1 and EVI1, whose deregulation is also known to be associated with MDS. RUNX1 is a master regulator of hematopoiesis that specifically binds to the DNA and regulates the expression of hematopoietic genes. Lately it has become clear that RUNX1 is frequently mutated in patients with MDS. Surprisingly, the mutations are limited to one allele, suggesting that a partial loss of function is sufficient to contribute to the disease. The second gene, EVI1, encodes a large nuclear protein characterized by two domains of zinc fingers. While the precise role of EVI1 is not known, this protein is not expressed in normal bone marrow and is inappropriately activated in very aggressive MDS by chromosomal translocations. Because alterations of both genes are not usually observed together, we have recently investigated whether there could be a potential link between the pathways regulated by RUNX1 and EVI1. Here, we report that in vivo and in vitro EVI1 physically interacts with RUNX1. We mapped the interaction domains to the eighth zinc finger motif of EVI1 and to the DNA-binding Runt domain of RUNX1. We found that this interaction alters the ability of RUNX1 to bind to its DNA consensus and represses the ability of RUNX1 to activate a reporter gene, suggesting that the interaction between EVI1 and RUNX1 could impair the hematopoietic programs regulated by RUNX1. These data suggest that inappropriate expression of EVI1 could contribute to hematopoietic transformation in part by a new mechanism that involves EVI1 association with key hematopoietic regulators leading to their functional impairment. Our results also indicate that the interaction region between RUNX1 and EVI1 could represent a novel target for therapeutic drugs for EVI1-associated leukemia.

RUNX1 also known as AML1 is a transcription factor essential for normal hematopoiesis, platelet production and thymocyte development. RUNX1 is the most frequent target of chromosomal translocations and acquired or inherited point mutations associated with human leukemia. RUNX1 is a DNA-binding transcription factor that can act both as an activator and a repressor of gene expression depending probably on the association of RUNX1 with co-activator or corepressors in large transcription complexes at promoter sites. The C-terminus of RUNX1 contains an inhibitory region, ID, which represses positive regulation of RUNX1-dependent genes. Thus, this region could potentially act as a switch and co-operate with RUNX1-interacting transcription factors in the choice between gene activation or gene repression. Here we have examined the role of ID in gene regulation by RUNX1. We found that this region is a homo-dimerization motif that controls RUNX1-RUNX1 interaction in vitro and in vivo. The association of RUNX1 with itself through this domain appears to reduce the positive transactivating potential of RUNX1 and, if provided in trans, this domain can repress by itself the activity of RUNX1 in reporter gene assays. Our studies suggest that RUNX1 autoregulates itself through its ability to form a homodimer. Data will be shown on the effects of the ID region in hematopoietic differentiation of cell lines. The results indicate that the ID region plays critical role in RUNX1 activity and is essential to control the correct execution of hematopoietic programs. It is of importance that aside from the t(12;21), all chromosomal translocations and virtually all point mutations associated with leukemia profoundly affect the integrity of the C-terminus including the ID region. These studies suggest a novel pathway involved in RUNX1 leukemogenesis and provide new targets for the management of RUNX1-leukemia. Kislay Sinha and Donglan Li contributed equally to this study.