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Spatiotemporal Uncoupling of MicroRNA-Mediated Translational Repression and Target RNA Degradation Controls MicroRNP Recycling in Mammalian Cells
No full textMicroRNA (miRNA)-mediated repression controls expression of more than half of protein-coding genes in metazoan animals. Translation repression is associated with target mRNA degradation initiated by decapping and deadenylation of the repressed mRNAs. Earlier evidence suggests the endoplasmic reticulum (ER) as the site where microRNPs (miRNPs) interact with their targets before translation repression sets in, but the subcellular location of subsequent degradation of miRNArepressed messages is largely unidentified. Here, we explore the subcellular distribution of essential components of degradation machineries of miRNA-targeted mRNAs. We have noted that interaction of target mRNAs with AGO2 protein on the ER precedes the relocalization of repressed messages to multivesicular bodies (MVBs). The repressed messages subsequently get deadenylated, lose their interaction with AGO2, and become decapped. Blocking maturation of endosomes to late endosome and MVBs by targeting the endosomal protein HRS uncouples miRNA-mediated
translation repression from target RNA degradation. HRS is also targeted by the intracellular parasite Leishmania donovani, which curtails the HRS level in infected cells
to prevent uncoupling of mRNA-AGO2 interaction, preventing degradation of translationally repressed messages, and thus stops recycling of miRNPs preengaged in repressio
Targeted apoptosis in ovarian cancer cells through mitochondrial dysfunction in response to Sambucus nigra agglutinin
Get PDFOvarian carcinoma (OC) patients encounter the severe challenge of clinical management owing to lack of screening measures, chemoresistance and finally dearth of non-toxic therapeutics. Cancer cells deploy various defense strategies to sustain the tumor microenvironment, among which deregulated apoptosis remains a versatile promoter of cancer progression. Although recent research has focused on identifying agents capable of inducing apoptosis in cancer cells, yet molecules efficiently breaching their
survival advantage are yet to be classified. Here we identify lectin, Sambucus nigra agglutinin (SNA) to exhibit selectivity towards identifying OC by virtue of its specific recognition of α-2, 6-linked sialic acids. Superficial binding of SNA to the OC cells confirm
the hyper-sialylated status of the disease. Further, SNA activates the signaling pathways of AKT and ERK1/2, which eventually promotes de-phosphorylation of dynamin-related protein-1 (Drp-1). Upon its translocation to the mitochondrial fission loci Drp-1 mediates the central role of switch in the mitochondrial phenotype to attain fragmented morphology. We confirmed mitochondrial
outer membrane permeabilization resulting in ROS generation and cytochrome-c release into the cytosol. SNA response resulted in an allied shift of the bioenergetics profile from Warburg phenotype to elevated mitochondrial oxidative phosphorylation, altogether highlighting the involvement of mitochondrial dysfunction in restraining cancer progression. Inability to replenish the SNA-induced energy crunch of the proliferating cancer cells on the event of perturbed respiratory outcome resulted in cell cycle
arrest before G2/M phase. Our findings position SNA at a crucial juncture where it proves to be a promising candidate for impeding progression of OC. Altogether we unveil the novel aspect of identifying natural molecules harboring the inherent capability of targeting mitochondrial structural dynamics, to hold the future for developing non-toxic therapeutics for treating OC
Transcriptional regulation in mitosis and its implications in genomic instability and cancer
No full textCancer is a multifactorial multistage human disease with the highest morbidity worldwide. Aneuploidy climaxing in genomic instability is a leading hallmark of cancer
progression. Alterations in mitotic pathways lead to aneuploidization of human cells. Proper progression of mitosis depends on orderly ubiquitination and subsequent degradation ofvarious mitotic inhibitors. At molecular level, Cdc20 activates E3 ubiquitin ligase Anaphase
Promoting Complex/Cyclosome (APC/C) which along with E2 ubiquitin conjugating enzyme, UbcH10, executes the function of mitotic progression. APC/CCDC20 is negatively regulated by spindle assembly checkpoint (SAC) that ensures accurate segregation of chromosomes during mitosis. SAC comprises MAD and BUB family of proteins which sense lack of tension at improperly attached or unattached kinetochores of metaphase chromosomes. Upon prevalence of errors in kinetochore-spindle attachment, SAC assembles at the unattached kinetochores and generates a ―wait anaphase‖ signal. At the molecular level, an activated SAC sequesters Cdc20 into an inhibitory complex known as Mitotic Checkpoint Complex (MCC) which inhibits APC/C and pauses cells in mitosis till the kinetochore attachment errors get rectified. Upon amphitellic kinetochore-spindle attachment, MCC is disassembled by APC/C-UbcH10 mediated ubiquitination following which Cdc20 associates with APC/C and catalyses mitotic progression of cells. UbcH10 thus plays a pivotal role in mitotic regulation. Interestingly, UbcH10 is found to be one of the majorly deregulated proteins in most cancer types with no reported mutation or polymorphisms. Thus functional regulation of UbcH10 through
proteasomal degradation is well studied but its transcriptional regulation is scarcely touched upon. The key role of UbcH10 in mitotic progression thus triggers interest in studying its transcriptional regulation in intricate details. Previous work from our lab reported a novel transcriptional regulatory role of Cdc20. Cdc20-APC/C-CBP complex upregulates UbcH10 through E2F1-DP1 heterodimer. E2F1 was observed to regulate UbcH10 expression in cell cycle dependent manner. Interestingly, Retinoblastoma (Rb) was observed to antagonize UbcH10
expresion. Rb and E2F1 exhibited mutually exclusive regulation of UbcH10 in G1 and G2/M phases of cell cycle respectively. Further, deregulated Rb-E2F1 pathway, through altered UbcH10 expression, contributed significantly to premature mitosis culminating in chromosomal errors and genomic instability. We thus explored a new facet of Rb functionality-its role in regulation of mitosis. UBCH10 expression is turned off during G1 phase. Rb, the master tumour suppressor, plays a pivotal role in this respect. Rb mediates its function by suppression of UbcH10 expression in G1 phase through the suppressive E2F family member-E2F4.The dissertation has explored that there is a novel dual suppressive complex involving Rb-E2F4 and DREAM which mediates UBCH10 repression. Rb and E2F4 collaborate to suppress UbcH10 expression through chromatin modifications. The negative control of UbcH10 monitors cellular
proliferation which gets significantly enhanced in cancer progression due to inactivation of Rb. We further explored the novel chromatin modifying role of Anaphase promoting
complex/Cyclosome(APC/C) .APC/C was found to modulate UbcH10 expression in G2/M phase through alteration in ubiquitination status of histone 2B. .H2B ubiquitination in
conjuction with altered H3 acetylation changes the chromatin dynamics .The chromatin alterations hence potentially help in driving the expression of UbcH10 and has strong implications in monitoring of temporal mitotic division of duplicated chromosomes. Thus the thesis aims to explore the mechanistic regulation of mitosis through transcriptional pathwaysusing UbcH10 as a model gene of study
Formation of Annular Protofibrillar Assembly by Cysteine Tripeptide: Unraveling the Interactions with NMR, FTIR, and Molecular Dynamics
No full textBoth hydrogen-bonding and hydrophobic interactions play a significant role in molecular assembly,including self-assembly of proteins and peptides. In this study,
we report the formation of annular protofibrillar structure (diameter ∼500 nm) made of a newly synthesized s-benzylprotected cysteine tripeptide, which was primarily stabilized by hydrogen-bonding and hydrophobic interactions. Atomic force microscopy and field emission scanning electron microscopy analyses found small oligomers (diameter ∼60 nm) to bigger annular (outer diameter ∼300 nm; inner diameter, 100 nm) and protofibrillar structures after 1−2 days of incubation.
Rotating-frame Overhauser spectroscopic (ROESY) analysis
revealed the presence of several nonbonded proton−proton
interactions among the residues, such as amide protons with methylene group, aromatic protons with tertiary butyl group, and methylene protons with tertiary butyl group. These added significant stability to bring the peptides closer to form a well-ordered assembled structure. Hydrogen−deuterium exchange NMR measurement further suggested that two individual amide protons among the three amide groups were strongly engaged with the adjacent tripeptide via H-bond interaction. However, the
remaining amide proton was found to be exposed to solvent and remained noninteracting with other tripeptide molecules. In addition to chemical shift values, a significant change in amide bond vibrations of the tripeptide was found due to the formation of the self-assembled structure. The amide I mode of vibrations involving two amide linkages appeared at 1641 and 1695 cm−1 in the solid state. However, in the assembled state, the stretching band at 1695 cm−1 became broad and slightly shifted to ∼1689 cm−1. On the contrary, the band at 1641 cm−1 shifted to 1659 cm−1 and indicated that the −CO bond associated with this vibration became stronger in the assembled state. These changes in Fourier transform infrared spectroscopy frequency clearly
indicated changes in the amide backbone conformation and the associated hydrogen-bonding pattern due to the formation of the assembled structure. In addition to hydrogen bonding, molecular dynamics simulation indicated that the number of π−π interactions also increased with increasing number of tripeptides participated in the self-assembly process. Combined results envisaged a cross β-sheet assembly unit consisting of four intermolecular hydrogen bonds. Such noncovalent peptide assemblies
glued by hydrogen-bonding and other weak forces may be useful in developing nanocapsule and related materials
Photo-induced cytotoxicity and anti-metastatic activity of ruthenium(II)–polypyridyl complexes functionalized with tyrosine or tryptophan
No full textThe synergistic effect of oxygen, light, and photosensitizer (PS) has found applications in medicine for the treatment of cancer through photodynamic therapy (PDT). Induction of apoptosis to cancerous cells will prevent tumor metastasis that spreads cancer cells to the neighboring organs/tissues. Herein, we report the two apoptotic Ru(II)–polypyridyl complexes that are functionalized with pendant amino acid moieties tyrosine (1) and tryptophan (2), respectively. These two water soluble complexes were found to interact strongly (K1a = (1.18 ± 0.28) × 105 M−1 and K2a = (1.57 ± 0.77) × 105 M−1) with CT-DNA. Isothermal titration calorimetry (ITC) studies revealed that these complexes bind to CT-DNA through an entropically driven process. Both the complexes showed photo-induced cytotoxicity and exhibit apoptotic activity under photo-irradiation conditions. The comet assay indicated that these complexes can damage cellular DNA,
which is attributed to the significant build-up of 1O2 level even on irradiation with low intensity light
(10 J cm−2, λRange 450–480 nm). This photoinduced DNA damage and apoptosis in A549 cells was induced by reactive oxygen species (ROS) and occurred through up-regulation of apoptotic marker caspase-3. Control experiments under dark conditions revealed an insignificant cytotoxicity towards
these cells for two photosensitive molecule
Stearylamine-Bearing Cationic Liposome For Targeted Anti-Cancer Therapy And Its Role In Immunomodulation
Get PDFEXPLORING THE UNIQUE PROTEOME OF LEISHMANIA DONOVANI FOR THE DEVELOPMENT OF NOVEL DRUG TARGETS AND VACCINE CANDIDATES:LEISHMANIA DONOVANI AURORA KINASES
No full textInteraction Of miRNP Machinery With Subcellular Structures And Organelles: Implications In miRNA Mediated Gene Repression Process In Mammalian Cells
No full textAny event inside the cell is determined by the synergistic functioning of
required proteins in a given time and space. Such spatiotemporal regulation is
important for cells, granting them ability to respond successfully to the plethora of
changes happening in their microenvironment in order to survive. Genes encode
proteins and proteins dictate cellular functions. Information flows from DNA to RNA
to protein, according to the central dogma of molecular biology and each step is
under stringent regulatory control to ensure cell fate and function. DNA or the cryptic
code of life is first copied into small messages as messenger RNA (mRNA)
molecules through transcription and then decryptified to the effector form as proteins
by translation. Cells can control where, when and how much of a particular gene is
transcribed and thereby translated. Regulation of these two major steps,
transcription and translation is critical for the adaptability of the cell. Regulation of
transcription and translation occurs in both prokaryotes and eukaryotes, but it is far
more complex in eukaryotes.
MicroRNA, the 22-24 nucleotides long non-coding RNA, forms miRNARibonucleoprotein
(miRNP) complex with Argonaute (Ago) proteins and posttranscriptionally
regulate diverse biological processes in metazoan cells. miRNA
mediated gene repression process occurs via either translation inhibition or mRNA
degradation (Bartel, 2009). Information on the subcellular sites where the miRNAmediated
repression happens is still profoundly limited. Recently, several studies in
plants as well as animals have established the association of miRNP complex with endomembranes during miRNA biogenesis and repressive function (Barman and
Bhattacharyya, 2015; Kim et al., 2014). In particular, endoplasmic reticulum (ER) has
been reported as the site for RISC (RNA induced silencing complex) nucleation as
well as translational repression whereas, RISC accumulation and possible action is
shown to be associated with endosomes and Multi-Vesicular Bodies (MVBs)
(Gibbings et al., 2009; Lee et al., 2009; Li et al., 2013; Rogers and Chen, 2013;
Stalder et al., 2013). So far none of the studies have linked miRNA based processes
happening on ER and endosomal compartments and a lacuna still remains in
understanding of ER and Endosome associated miRNA turnover. Other key
organelles like mitochondria, primarily considered as power-houses in eukaryotic
cells, show enrichment for a specific subset of miRNAs (Bandiera et al., 2011).
Additionally, effect of mitochondrial membrane potential disruption on RNA
interference and dynamics of cytoplasmic RNA granules such as Processing-body
(P-body) have been documented in mammalian cells (Huang et al., 2011a; Kren et
al., 2009). Cumulatively, these evidences indicate towards a spatial as well as
temporal association of miRNPs with different organelles. But how these subcellular
pools of miRNP complexes interact and exchange their components has not been
addressed before. This phenomenon has potential role in modulation of miRNA
activity and stability.
miRNAs are relatively stable molecules with predominantly slow rate of
turnover in animal cells (Carrion et al., 2013; Fabian et al., 2010; Gregory et al.,
2008). For ensuring rapid switching between subcellular miRNA repertoire, enabling
a single miRNA to repress multiple targets (Hutvagner and Zamore, 2002; Mourelatos et al., 2002), recycling of miRNP is essential. But, how these organellar
pools of miRNAs, complexed with Ago proteins get replaced with new miRNAs to
target different sets of genes is largely unknown. Specific changes in cellular miRNA
profile under altered cellular environment have been reported. Many human
diseases are associated with defective miRNA mediated repression of target mRNAs
(Mendell and Olson, 2012). Multitudes of pathogenic parasites are known to alter the
miRNA mediated repression process to their own advantage (Huang et al., 2011b;
Manzano-Roman and Siles-Lucas, 2012; Skalsky and Cullen, 2010). Leishmania
donovani (Ld), is a dimorphic pathogenic parasite which causes visceral
leishmaniasis or kala-azar in vertebrates (Murray et al., 2005). Upon its entry through
sand fly bite (Desjardins and Descoteaux, 1998; Engwerda et al., 2004; Olivier et al.,
2005) for efficient infection of macrophages, Ld downregulates the pro-inflammatory
response of host macrophages through induced expression of the Uncoupler Protein
2 (Ucp2) (Basu Ball et. al., 2011; Rousset et al., 2006), a mitochondrial membrane
protein which modulates mitochondrial membrane potential by uncoupling the
electron transport chain (Skulachev, 1998).
In this study it has been reported that the pathogen Leishmania donovani
invades the host macrophage and induces expression of Ucp2 and downregulates
Mitofusin (Mfn) 2 to depolarize mitochondria and detether endosomes from ER
causing defect in miRNP recycling. This miRNP recycling is essential to maintain the
miRNA-AGO2 (miRISC) complex homeostatic levels by controlling the subcellular
distribution of miRNA biogenesis associated factors and their interaction with each
other. Moreover, this loss of functionality of mitochondrial membrane potential (ΔΨM) sensitive mitochondria-ER juxtapositioning also regulates P-body biogenesis by affecting the miRNA repression process. Furthermore, this phenomenon is also
mimicked in high density culture based physiological paradigm. Mfn1 and Mfn2 are
mitochondrial dynamin related GTPases. Global ablation of both Mfn1 and Mfn2
gene results in embryonic death. Remarkably, with conditional knockout crerecombinase
system although the mice are born alive but, only the mice ubiquitously
lacking MFN1 are apparently healthy, whereas mice without MFN2 die in the early
postnatal period as well as show severe defects (Chen et al., 2003; Chen et al.,
2007). Mfn2 with its lower GTPase activity is present on both mitochondrial and ER
membranes and plays a key role in stabilizing ER-mitochondria tethering apart from
its well-established role in mitochondrial fusion (Chang et al., 2004; de Brito and
Scorrano, 2008; Ishihara et al., 2004; Schrepfer and Scorrano, 2016). Mfn2 ablation
or depletion results in reduced colocalisation of ER with endosomes. Further
experimentation with non-macrophage cells, allowed us to document reduced
recycling of existing miRNP complexes and curtailed rate of miRNP biogenesis due
to mitochondria-ER de-tethering. Thus, a unique mode of post-transcriptional gene
regulation was identified in mammalian cells where inter-organellar interaction could
control turnover and activity of cellular miRNA-Ago complex. This process is affected
by Ld to alter miRNA activity during the infection of host macrophage cells that
restricts production of pro-inflammatory cytokines
Understanding Extracellular Matrix Remodelling in Rheumatic Heart Valve for Biomarker Identification
Get PDFSelective Recognition of H3.1K36 Dimethylation/H4K16 Acetylation Facilitates the Regulation of All-trans-retinoic Acid (ATRA)-responsive Genes by Putative Chromatin Reader ZMYND8
No full textZMYND8 (zinc finger MYND (Myeloid, Nervy and DEAF-1)-
type containing 8), a newly identified component of the transcriptional coregulator network, was found to interact with the Nucleosome Remodeling and Deacetylase (NuRD) complex. Previous reports have shown that ZMYND8 is instrumental in recruiting the NuRD complex to damaged chromatin for repressing transcription and promoting double strand break repair by homologous recombination. However, the mode of transcription regulation byZMYND8has remained elusive. Here, we report that through its specific key residues present in its conserved chromatin-
binding modules,ZMYND8interacts with the selective epigenetic marks H3.1K36Me2/H4K16Ac. Furthermore, ZMYND8
shows a clear preference for canonical histone H3.1 over variant H3.3. Interestingly, ZMYND8 was found to be recruited to several developmental genes, including the all-trans-retinoic acid (ATRA)-responsive ones, through its modified histone-binding ability. Being itself inducible byATRA,this zinc finger transcription factor is involved in modulating other ATRA-inducible genes. We found
that ZMYND8 interacts with transcription initiation-competent RNA polymerase II phosphorylated at Ser-5 in a DNA templatedependent manner and can alter the global gene transcription. Overall, our study identifies that ZMYND8 has CHD4-independent functions in regulating gene expression through its modified histone-binding ability