Publications by authors named "Mohd Saleem Dar"

13 Publications

  • Page 1 of 1

Transcriptome-wide identification of squalene epoxidase genes from Glycyrrhiza glabra L.: expression analysis and heterologous expression of GgSQE1 suggest important role in terpenoid biosynthesis.

Protoplasma 2021 Feb 24. Epub 2021 Feb 24.

Plant Biotechnology Division, CSIR-IIIM, Jammu, India.

Squalene epoxidase (SQE) is a crucial regulatory enzyme for the biosynthesis of several important classes of compounds including sterols and triterpenoids. The present paper identified and characterised five SQE genes (GgSQE1 to GgSQE5) from Glycyrrhiza glabra through transcriptome data mining and homology-based cloning, for the first time. The phylogenetic analysis implied their functional divergence. The ORF corresponding to one of the five SQEs, namely, GgSQE1, was cloned and studied for its function in a heterologous system, following transient and stable expressions. The transient expression followed by GgSQE1 encoding protein purification suggested approximately 58.0-kDa protein following the predicted molecular mass of the deduced protein. The gene expression profiling based on qRT-PCR indicated its highest expression (6.4-folds) in the 10-month-old roots. Furthermore, ABA (12.4-folds) and GA (2.47) treatments upregulated the expression of GgSQE1 in the shoots after 10 and 12 hours, respectively, which was also reflected in glycyrrhizin accumulation. The inductive effects of ABA and GA over GgSQE1 expression were also confirmed through functional analysis of GgSQE1 promoters using GUS fusion construct. Stable constitutive expression of GgSQE1 in Nicotiana tabacum modulated the sterol contents. The study could pave the way for understanding the metabolic flux regulation concerning biosynthesis of related sterols and triterpenoids.
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http://dx.doi.org/10.1007/s00709-021-01616-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7904238PMC
February 2021

Oct4-Mediated Inhibition of Lsd1 Activity Promotes the Active and Primed State of Pluripotency Enhancers.

Cell Rep 2020 02;30(5):1478-1490.e6

Department of Biochemistry, Purdue University, West Lafayette, IN 47907, USA; Purdue University Center for Cancer Research, Purdue University, West Lafayette, IN 47907, USA. Electronic address:

An aberrant increase in pluripotency gene (PpG) expression due to enhancer reactivation could induce stemness and enhance the tumorigenicity of cancer stem cells. Silencing of PpG enhancers (PpGe) during embryonic stem cell differentiation involves Lsd1-mediated H3K4me1 demethylation and DNA methylation. Here, we observed retention of H3K4me1 and DNA hypomethylation at PpGe associated with a partial repression of PpGs in F9 embryonal carcinoma cells (ECCs) post-differentiation. H3K4me1 demethylation in F9 ECCs could not be rescued by Lsd1 overexpression. Given our observation that H3K4me1 demethylation is accompanied by strong Oct4 repression in P19 ECCs, we tested if Oct4 interaction with Lsd1 affects its catalytic activity. Our data show a dose-dependent inhibition of Lsd1 activity by Oct4 and retention of H3K4me1 at PpGe in Oct4-overexpressing P19 ECCs. These data suggest that Lsd1-Oct4 interaction in cancer stem cells could establish a "primed" enhancer state that is susceptible to reactivation, leading to aberrant PpG expression.
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http://dx.doi.org/10.1016/j.celrep.2019.11.040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7316367PMC
February 2020

Tweaking genome-editing approaches for virus interference in crop plants.

Plant Physiol Biochem 2020 Feb 19;147:242-250. Epub 2019 Dec 19.

Division of Plant Pathology, Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir, Shalimar, Srinagar, J&K, 190025, India.

Plant viruses infect various economically important crops and cause a serious threat to agriculture. As of now, conventional strategies employed are inadequate to circumvent the proliferation of rapidly evolving plant viruses. In this regard, recent advancement in genome-editing approach looks promising to produce plants resistant to DNA/RNA virus infections. Clustered regularly interspaced palindromic repeats (CRISPR) system has been emerged as a promising genome-editing tool that has received special interest because of its ease, competence and reproducibility. Recent studies have demonstrated that CRISPR/Cas9 system has great potential to confer plant immunity by either directly targeting or cleaving the viral genome in both RNA and DNA viruses. Similarly, the approach can be used for targeting the host susceptibility genes more particularly in case of RNA viruses. In the present review, different approaches and strategies being used to improve plant resistance against devastating viruses are discussed in view of recent advances in CRISPR systems. This review also describes the major pitfalls of CRISPR/Cas9 system that utilizes highly efficient and novel platforms to engineer interference to single and multiple plant RNA viruses.
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http://dx.doi.org/10.1016/j.plaphy.2019.12.022DOI Listing
February 2020

Identification of dinactin, a macrolide antibiotic, as a natural product-based small molecule targeting Wnt/β-catenin signaling pathway in cancer cells.

Cancer Chemother Pharmacol 2019 09 25;84(3):551-559. Epub 2019 May 25.

Academy of Scientific and Innovative Research, New Delhi, India.

Purpose: Despite the fact that hyper-activation of Wnt/β-catenin signaling pathway has been seen in many cancers, including liver, colorectal and lung carcinoma, no small molecule inhibitors are available that specifically target this pathway. In this study, we analyzed the impact of dinactin (DA), an antibiotic ionophore produced by Streptomyces species, as an effective small molecule targeting Wnt/β-catenin signaling pathway in cancer cells.

Methods: We performed MTT assays to investigate cell viability and proliferation after exposure to small molecules. Protein expression analysis was carried out by western blotting. Top-Flash reporter assays were used to score for β-catenin signaling and cell cycle analysis was carried out by flow cytometry.

Results: In the first set of experiments, DA was seen to selectively inhibit the proliferation of HCT-116 and HepG2 cancer cells, unlike HEK-293 cells (a low tumorigenic cell line), in apoptosis-independent manner. Further, DA was seen to block the G1/S progression and decrease the expression of cyclin D1 in cancer cells. Since cyclin D1 is the downstream target gene of Wnt/β-catenin signaling, we examined the impact of DA on TCF-dependent β-catenin activity using Top-Flash reporter assay. Interestingly, DA significantly decreased Top-Flash activity at lower nano-molar concentrations when compared with salinomycin in HCT-116 and HepG2 cells.

Conclusion: We report the identification of dinactin as a natural product-based small molecule that effectively blocks the Wnt/β-catenin signaling pathway in cancer cells at nano-molar concentration. We anticipate that DA could be developed as a novel drug for anti-cancer therapy and for the management of neuropathic pain.
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http://dx.doi.org/10.1007/s00280-019-03870-xDOI Listing
September 2019

Effect of Disease-Associated Germline Mutations on Structure Function Relationship of DNA Methyltransferases.

Genes (Basel) 2019 05 14;10(5). Epub 2019 May 14.

Department of Biochemistry, Purdue University, West Lafayette, IN 47907, USA.

Despite a large body of evidence supporting the role of aberrant DNA methylation in etiology of several human diseases, the fundamental mechanisms that regulate the activity of mammalian DNA methyltransferases (DNMTs) are not fully understood. Recent advances in whole genome association studies have helped identify mutations and genetic alterations of DNMTs in various diseases that have a potential to affect the biological function and activity of these enzymes. Several of these mutations are germline-transmitted and associated with a number of hereditary disorders, which are potentially caused by aberrant DNA methylation patterns in the regulatory compartments of the genome. These hereditary disorders usually cause neurological dysfunction, growth defects, and inherited cancers. Biochemical and biological characterization of DNMT variants can reveal the molecular mechanism of these enzymes and give insights on their specific functions. In this review, we introduce roles and regulation of DNA methylation and DNMTs. We discuss DNMT mutations that are associated with rare diseases, the characterized effects of these mutations on enzyme activity and provide insights on their potential effects based on the known crystal structure of these proteins.
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http://dx.doi.org/10.3390/genes10050369DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6562416PMC
May 2019

Analyzing the role of cannabinoids as modulators of Wnt/β-catenin signaling pathway for their use in the management of neuropathic pain.

Bioorg Med Chem Lett 2019 05 11;29(9):1043-1046. Epub 2019 Mar 11.

Natural Product Chemistry Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu 180001, India; Academy of Scientific and Innovative Research, New Delhi, India. Electronic address:

Neuropathic pain is a debilitating form of treatment-resistant chronic pain caused by damage to the nervous system. Cannabinoids have been known for suppressing neuropathic pain by modulating the endo cannabinoid system. Since the canonical Wnt/β-catenin signaling has recently been implicated in pain sensation, we investigated the impact of major cannabinoids (1-6) from the leaves of Cannabis sativa and an epoxy derivative of compound 2, here upon referred to as 2a, on modulating Wnt/β-catenin signaling pathway. The results presented in this study show that compound 1, 2 and 2a exhibited potent inhibitory activity against Wnt/β-catenin pathway in a dose-dependent manner. Compound 2a was seen to inhibit this pathway at slightly lower concentrations than its parent molecule 2, under similar conditions. Taken together, compound 1, 2 and 2a, by virtue of their inhibition of Wnt/β-catenin signaling pathway, could be developed as effective neuroprotective agents for the management of neuropathic pain.
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http://dx.doi.org/10.1016/j.bmcl.2019.03.013DOI Listing
May 2019

Corrigendum to "Streptomyces puniceus strain AS13., Production, characterization and evaluation of bioactive metabolites: A new face of dinactin as an antitumor antibiotic" [Microbiological Research 207, (2018)196-202].

Microbiol Res 2018 12 9;217:108. Epub 2018 Oct 9.

Microbial Biotechnology Division, CSIR - Indian Institute of Integrative Medicine, Sanatnagar, Srinagar, Jammu & Kashmir, 190005, India; Academy of Scientific and Innovative Research, CSIR - Indian Institute of Integrative Medicine, Canal Road, Jammu Tawi, 180001, India. Electronic address:

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http://dx.doi.org/10.1016/j.micres.2018.10.001DOI Listing
December 2018

Identification of a unique loss-of-function mutation in IGF1R and a crosstalk between IGF1R and Wnt/β-catenin signaling pathways.

Biochim Biophys Acta Mol Cell Res 2018 Jun 3;1865(6):920-931. Epub 2018 Apr 3.

Academy of Scientific and Innovative Research (AcSIR), Anusandhan Bhawan, New Delhi, India; Cancer Pharmacology Division, CSIR-Indian Institute of Integrative Medicine, Jammu 180001, Jammu & Kashmir, India. Electronic address:

IGF1R is a ubiquitous receptor tyrosine kinase that plays critical roles in cell proliferation, growth and survival. Clinical studies have demonstrated upregulation of IGF1R mediated signaling in a number of malignancies including colon, breast, and lung cancers. Overexpression of the IGF1R in these malignancies is associated with a poor prognosis and overall survival. IGF1R specific kinase inhibitors have failed in multiple clinical trials partly because of the complex nature of IGF1R signaling. Thus identifying new binding partners and allosteric sites on IGF1R are emerging areas of research. More recently, IGF1R has been shown to translocate into the nucleus and perform many functions. In this study, we generated a library of IGF1R deletion and point mutants to examine IGF1R subcellular localization and activation of downstream signaling pathways. We show that the nuclear localization of IGF1R is primarily defined by its cytoplasmic domain. We identified a cross-talk between IGF1R and Wnt/β-catenin signaling pathways and showed, for the first time, that IGF1R is associated with upregulation of TCF-mediated β-catenin transcriptional activity. Using loss-of-function mutants, deletion analysis and IGF1R specific inhibitor(s), we show that cytoplasmic and nuclear activities are two independent functions of IGF1R. Furthermore, we identified a unique loss-of-function mutation in IGF1R. This unique loss-of-function mutant retains only nuclear functions and sits in a pocket, outside ATP and substrate binding region, that is suited for designing allosteric inhibitors of IGF1R.
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http://dx.doi.org/10.1016/j.bbamcr.2018.03.013DOI Listing
June 2018

Streptomyces puniceus strain AS13., Production, characterization and evaluation of bioactive metabolites: A new face of dinactin as an antitumor antibiotic.

Microbiol Res 2018 03 8;207:196-202. Epub 2017 Dec 8.

Microbial Biotechnology Division, CSIR - Indian Institute of Integrative Medicine, Sanatnagar, Srinagar, Jammu & Kashmir, 190005, India; Academy of Scientific and Innovative Research, CSIR - Indian Institute of Integrative Medicine, Canal Road, Jammu Tawi, 180001, India. Electronic address:

A highly active actinobacterial strain isolated from untapped areas of Northwestern Himalayas and characterised as Streptomyces puniceus strain AS13 by 16S rRNA gene sequencing was selected for production of bioactive metabolites. The bioassay-guided fractionation of microbial cultured ethyl acetate extract of the strain, led to isolation of macrotetrolide compound 1 (Dinactin) and compound 2 (1-(2,4-dihydroxy-6-methylphenyl)-ethanone). Structures of the isolated compounds were elucidated by [corrected] interpretation of NMR and other spectroscopic data including HR-ESI-MS, FT-IR. These compounds are reported for first time from Streptomyces Puniceus. Compound 1 exhibited strong anti-microbial activity against all tested bacterial pathogens including Mycobacterium tuberculosis. The MIC values of compound 1 against Gram negative and Gram positive bacterial pathogens ranged between 0.019 - 0.156μgml and 1μgml against Mycobacterium tuberculosis H37Rv. Dinactin exhibited marked anti-tumor potential with IC of 1.1- 9.7μM in various human cancerous cell lines and showed least cytotoxicity (IC∼80μM) in normal cells (HEK-293). Dinactin inhabited cellular proliferation in cancer cells, reduced their clonogenic survival as validated by clonogenic assay and also inhabited cell migration and invasion characteristics in colon cancer (HCT-116) cells. Our results expressed the antimicrobial potential of dinactin and also spotted its prospective as an antitumor antibiotic.
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http://dx.doi.org/10.1016/j.micres.2017.12.004DOI Listing
March 2018

Βeta-catenin N-terminal domain: An enigmatic region prone to cancer causing mutations.

Mutat Res 2017 07 12;773:122-133. Epub 2017 Jun 12.

Academy of Scientific and Innovative Research (AcSIR), New Delhi, India; Cancer Pharmacology Division, CSIR-Indian Institute of Integrative Medicine, Jammu, J&K, India. Electronic address:

The Wnt/β-catenin is a highly conserved signaling pathway involved in cell fate decisions during various stages of development. Dysregulation of canonical Wnt/β-catenin signaling has been associated with various diseases including cancer. β-Catenin, the central component of canonical Wnt signaling pathway, is a multi-functional protein playing both structural and signaling roles. β-Catenin is composed of three distinct domains: N-terminal domain, C-terminal domain and a central armadillo repeat domain. N-terminal domain of β-catenin harbours almost all of the cancer causing mutations, thus deciphering its critical structural and functional roles offers great potential in cancer detection and therapy. Here, in this review, we have collected information from pharmacological analysis, bio-physical and structural studies, molecular modeling, in-vivo and in-vitro assays, and transgenic animal experiments employing various N-terminal domain variants of β-catenin to discuss the interaction of β-catenin with its binding partners that specifically interact with this domain and the implications of these interactions on signaling, cell fate determination, and in tumorigenesis. A thorough understanding of interactions between β-catenin and its binding partners will enable us to more effectively understand how β-catenin switches between its multiple roles, and will lead to the development of specific assays for the identification of small molecules as chemotherapeutic agents to treat diseases, including cancer and neurological disorders, where Wnt/β-catenin signaling is dysregulated.
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http://dx.doi.org/10.1016/j.mrrev.2017.06.001DOI Listing
July 2017

p110α and p110β isoforms of PI3K signaling: are they two sides of the same coin?

FEBS Lett 2016 09 12;590(18):3071-82. Epub 2016 Sep 12.

Academy of Scientific and Innovative Research, New Delhi, India.

Class-1 phosphatidylinositol-3-kinases (PI3Ks) are activated by a variety of extracellular stimuli and have been implicated in a wide range of cellular processes. p110α and p110β are the two most studied isoforms of the class-1A PI3K signaling pathway. Although these two isoforms are ubiquitously expressed and play multiple redundant roles, they also have distinct functions within the cell. More recently, p110α and p110β isoforms have been shown to translocate into the nucleus and play a role in DNA replication and repair, and in cell cycle progression. In the following Review article, we discuss the overlapping and unique roles of p110α and p110β isoforms with a particular focus on their structure, expression analysis, subcellular localization, and signaling contributions in various cell types and model organisms.
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http://dx.doi.org/10.1002/1873-3468.12377DOI Listing
September 2016

Terminal regions of β-catenin are critical for regulating its adhesion and transcription functions.

Biochim Biophys Acta 2016 09 29;1863(9):2345-57. Epub 2016 Jun 29.

Academy of Scientific and Innovative Research (AcSIR), New Delhi, India; Cancer Pharmacology Division, CSIR-Indian Institute of Integrative Medicine, Jammu, J&K, India. Electronic address:

β-Catenin, the central molecule of canonical Wnt signaling pathway, has multiple binding partners and performs many roles in the cell. Apart from being a transcriptional activator, β-catenin acts as a crucial effector component of cadherin/catenin complex to physically interact with actin cytoskeleton along with α-catenin and E-cadherin for regulating cell-cell adhesion. Here, we have generated a library of β-catenin point and deletion mutants to delineate regions within β-catenin that are important for α-catenin-β-catenin interaction, nuclear localization, and transcriptional activity of β-catenin. We observed a unique mechanism for nuclear localization of β-catenin and its mutants and show that N-terminal exon-3 region and C-terminal domain of β-catenin are critical for this activity of β-catenin. Furthermore, we show HepG2 cells have high β-catenin mediated transcriptional activity due to the presence of an interstitial deletion at the N-terminal region of β-catenin. Due to this deletion mutant (hereupon called TM), GSK3β and HDAC inhibitors failed to show any impact whereas curcumin significantly inhibited β-catenin mediated transcriptional activity reiterating that TM is primarily responsible for the high transcriptional activity of HepG2 cells. Moreover, we show the recombinant TM does not physically interact with α-catenin, localizes predominantly in the nucleus, and has nearly two-fold higher transcriptional activity than the wildtype β-catenin.
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http://dx.doi.org/10.1016/j.bbamcr.2016.06.010DOI Listing
September 2016

Dynamics of GFP-Fusion p110α and p110β Isoforms of PI3K Signaling Pathway in Normal and Cancer Cells.

J Cell Biochem 2016 12 3;117(12):2864-2874. Epub 2016 Jun 3.

Academy of Scientific and Innovative Research, New Delhi, India.

Cancer therapeutics is a hot subject and PI3K class 1A isoforms (p110α and p110β) are pursued as major targets. Genetic analysis, biochemical approaches, and structural studies have demonstrated crucial roles for these isoforms in several physiological processes. p110α is critical for insulin signaling, whereas p110β is essential for the growth and differs from p110α in many ways. Here, we have generated GFP-fusion clones of wildtype and mutant version of p110α and p110β and expressed them in HEK293 and cancer cells to examine their subcellular localization and their impact on downstream signaling. In HEK293 cells, p110β GFP-fusion protein is translocated into the nucleus, whereas p110α-GFP stays exclusively in the cytoplasm. This study demonstrates that p110α and p110β oncogenecity, kinase activity, and interaction with p85 regulatory subunit does not have any impact on their subcellular localization. PI3K pathway specific inhibitor, LY294002, abrogated PI3K signaling by reducing pAkt levels, however, the subcellular localization of p110α and p110β remained unchanged. Furthermore, we analyzed the expression of recombinant p110α and p110β in a panel of human cancer cells and observed remarkable differences in their expression levels. The differential expression of recombinant p110α and p110β was observed to be mainly regulated by the endogenous levels of pAkt. Unlike in HEK293, p110α showed nuclear localization in cancer cells in a similar fashion to p110β. Moreover, we observed the PI3K signaling activities in low pAkt expressing cells are mediated by PDK1 and S6K proteins. Finally, p110α and p110β were seen to play an essential role in promoting the cell cycle progression in MCF-7 and HCT-116 cells. J. Cell. Biochem. 117: 2864-2874, 2016. © 2016 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/jcb.25598DOI Listing
December 2016