Publications by authors named "Ashraf Dar"

14 Publications

  • Page 1 of 1

Asymptomatic malaria infection prevailing risks for human health and malaria elimination.

Infect Genet Evol 2021 09 30;93:104987. Epub 2021 Jun 30.

Department of Biochemistry, School of Life Sciences, Central University of Rajasthan, Bandarsindri, Kishangarh, Ajmer, 305817, Rajasthan, India.

There has been a consistent rise in malaria cases in the last few years. The existing malaria control measures are challenged by insecticide resistance in the mosquito vector, drug résistance in parasite populations, and asymptomatic malaria (ASM) in healthy individuals. The absence of apparent malaria symptoms and the presence of low parasitemia makes ASM a hidden reservoir for malaria transmission and an impediment in malaria elimination efforts. This review focuses on ASM in malaria-endemic countries and the past and present research trends from those geographical locations. The harmful impacts of asymptomatic malaria on human health and its contribution to disease transmission are highlighted. We discuss certain crucial genetic changes in the parasite and host immune response necessary for maintaining low parasitemia leading to long-term parasite survival in the host. Since the chronic health effects and the potential roles for disease transmission of ASM remain mostly unknown to significant populations, we offer proposals for developing general awareness. We also suggest advanced technology-based diagnostic methods, and treatment strategies to eliminate ASM.
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http://dx.doi.org/10.1016/j.meegid.2021.104987DOI Listing
September 2021

The Deubiquitinase USP46 Is Essential for Proliferation and Tumor Growth of HPV-Transformed Cancers.

Mol Cell 2018 12 8;72(5):823-835.e5. Epub 2018 Nov 8.

Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, VA 22908, USA. Electronic address:

High-risk human papilloma viruses (HPVs) cause cervical, anal, and oropharyngeal cancers, unlike the low-risk HPVs, which cause benign lesions. E6 oncoproteins from the high-risk strains are essential for cell proliferation and transformation in HPV-induced cancers. We report that a cellular deubiquitinase, USP46, is selectively recruited by the E6 of high-risk, but not low-risk, HPV to deubiqutinate and stabilize Cdt2/DTL. Stabilization of Cdt2, a component of the CRL4 E3 ubiquitin ligase, limits the level of Set8, an epigenetic writer, and promotes cell proliferation. USP46 is essential for the proliferation of HPV-transformed cells, but not of cells without HPV. Cdt2 is elevated in human cervical cancers and knockdown of USP46 inhibits HPV-transformed tumor growth in xenografts. Recruitment of a cellular deubiquitinase to stabilize key cellular proteins is an important activity of oncogenic E6, and the importance of E6-USP46-Cdt2-Set8 pathway in HPV-induced cancers makes USP46 a target for the therapy of such cancers.
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http://dx.doi.org/10.1016/j.molcel.2018.09.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6294304PMC
December 2018

14-3-3 proteins play a role in the cell cycle by shielding cdt2 from ubiquitin-mediated degradation.

Mol Cell Biol 2014 Nov 25;34(21):4049-61. Epub 2014 Aug 25.

Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, Virginia, USA

Cdt2 is the substrate recognition adaptor of CRL4(Cdt2) E3 ubiquitin ligase complex and plays a pivotal role in the cell cycle by mediating the proteasomal degradation of Cdt1 (DNA replication licensing factor), p21 (cyclin-dependent kinase [CDK] inhibitor), and Set8 (histone methyltransferase) in S phase. Cdt2 itself is attenuated by SCF(FbxO11)-mediated proteasomal degradation. Here, we report that 14-3-3 adaptor proteins interact with Cdt2 phosphorylated at threonine 464 (T464) and shield it from polyubiquitination and consequent proteasomal degradation. Depletion of 14-3-3 proteins promotes the interaction of FbxO11 with Cdt2. Overexpressing 14-3-3 proteins shields Cdt2 that has a phospho-mimicking mutation (T464D [change of T to D at position 464]) but not Cdt2(T464A) from ubiquitination. Furthermore, the delay of the cell cycle in the G2/M phase and decrease in cell proliferation seen upon depletion of 14-3-3γ is partly due to the accumulation of the CRL4(Cdt2) substrate, Set8 methyltransferase. Therefore, the stabilization of Cdt2 is an important function of 14-3-3 proteins in cell cycle progression.
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http://dx.doi.org/10.1128/MCB.00838-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4386460PMC
November 2014

Potent antimalarial activity of acriflavine in vitro and in vivo.

ACS Chem Biol 2014 Oct 14;9(10):2366-73. Epub 2014 Aug 14.

Special Centre for Molecular Medicine, Jawaharlal Nehru University , New Delhi 110067, India.

Malaria continues to be a major health problem globally. There is an urgent need to find new antimalarials. Acriflavine (ACF) is known as an antibacterial agent and more recently as an anticancer agent. Here, we report that ACF inhibits the growth of asexual stages of both chloroquine (CQ) sensitive and resistant strains of human malarial parasite, Plasmodium falciparum in vitro at nanomolar concentration. ACF clears the malaria infection in vivo from the bloodstreams of mice infected with Plasmodium berghei. Interestingly, ACF is accumulated only in the parasitized red blood cells (RBCs) and parasite specific transporters may have role in this specific drug accumulation. We further show that ACF impairs DNA replication foci formation in the parasites and affects the enzymatic activities of apicoplast specific Gyrase protein. We thus establish ACF as a potential antimalarial amidst the widespread incidences of drug resistant Plasmodium strains.
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http://dx.doi.org/10.1021/cb500476qDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4201339PMC
October 2014

CRL4Cdt2 E3 ubiquitin ligase and proliferating cell nuclear antigen (PCNA) cooperate to degrade thymine DNA glycosylase in S phase.

J Biol Chem 2014 Aug 24;289(33):23056-23064. Epub 2014 Jun 24.

Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, Virginia 22908. Electronic address:

Thymine DNA glycosylase (TDG) is an essential enzyme playing multiple roles in base excision repair, transcription regulation, and DNA demethylation. TDG mediates the cytotoxicity of the anti-cancer chemotherapeutic drug 5-fluorouracil (5-FU) by prolonging S phase, generating DNA strand breaks, and inducing DNA damage signaling. During S phase of the cell cycle, TDG is degraded via the proteasomal pathway. Here we show that CRL4(Cdt2) E3 ubiquitin ligase promotes ubiquitination and proteasomal degradation of TDG in S phase in a reaction that is dependent on the interaction of TDG with proliferating cell nuclear antigen (PCNA). siRNA-mediated depletion of PCNA or components of CRL4(Cdt2), specifically cullin4A/B or substrate adaptor Cdt2, stabilizes TDG in human cells. Mutations in the PCNA-interacting peptide (PIP) motif of TDG that disrupt the interaction of TDG with PCNA or change critical basic residues essential for the action of the PIP degron prevent the ubiquitination and degradation of TDG. Thus physical interaction of TDG with PCNA through the PIP degron is required for targeting TDG to the CRL4(Cdt2) E3 ubiquitin ligase complex. Compared with forced expression of wild type TDG, CRL4(Cdt2)- resistant TDG (ΔPIP) slows cell proliferation and slightly increases the toxicity of 5-FU. Thus, CRL4(Cdt2)-dependent degradation of TDG occurs in S phase because of the requirement for TDG to interact with chromatin-loaded PCNA, and this degradation is important for preventing toxicity from excess TDG.
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http://dx.doi.org/10.1074/jbc.M114.574210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4132804PMC
August 2014

Deubiquitination of Tip60 by USP7 determines the activity of the p53-dependent apoptotic pathway.

Mol Cell Biol 2013 Aug 17;33(16):3309-20. Epub 2013 Jun 17.

Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, Virginia, USA.

Tip60 is an essential acetyltransferase required for acetylation of nucleosomal histones and other nonhistone proteins. Tip60 acetylates the p53 tumor suppressor at lysine 120 (K120), a modification essential for p53-dependent induction of PUMA and apoptosis. It is known that Tip60 is turned over in cells by the ubiquitin-proteasome system. However, the deubiquitinase activity for stabilizing Tip60 is unknown. Here we show that USP7 interacts with and deubiquitinates Tip60 both in vitro and in vivo. USP7 deubiquitinase activity is required for the stabilization of Tip60 in order to operate an effective p53-dependent apoptotic pathway in response to genotoxic stress. Inhibiting USP7 with the small-molecule inhibitor P22077 attenuates the p53-dependent apoptotic pathway by destabilizing Tip60. P22077, however, is still cytotoxic, and this is partly due to destabilization of Tip60.
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http://dx.doi.org/10.1128/MCB.00358-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3753911PMC
August 2013

Defective nuclear import of Tpr in Progeria reflects the Ran sensitivity of large cargo transport.

J Cell Biol 2013 May 6;201(4):541-57. Epub 2013 May 6.

Center for Cell Signaling, University of Virginia, Charlottesville, VA 22903, USA.

The RanGTPase acts as a master regulator of nucleocytoplasmic transport by controlling assembly and disassembly of nuclear transport complexes. RanGTP is required in the nucleus to release nuclear localization signal (NLS)-containing cargo from import receptors, and, under steady-state conditions, Ran is highly concentrated in the nucleus. We previously showed the nuclear/cytoplasmic Ran distribution is disrupted in Hutchinson-Gilford Progeria syndrome (HGPS) fibroblasts that express the Progerin form of lamin A, causing a major defect in nuclear import of the protein, translocated promoter region (Tpr). In this paper, we show that Tpr import was mediated by the most abundant import receptor, KPNA2, which binds the bipartite NLS in Tpr with nanomolar affinity. Analyses including NLS swapping revealed Progerin did not cause global inhibition of nuclear import. Rather, Progerin inhibited Tpr import because transport of large protein cargoes was sensitive to changes in the Ran nuclear/cytoplasmic distribution that occurred in HGPS. We propose that defective import of large protein complexes with important roles in nuclear function may contribute to disease-associated phenotypes in Progeria.
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http://dx.doi.org/10.1083/jcb.201212117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3653351PMC
May 2013

RVBs are required for assembling a functional TIP60 complex.

Mol Cell Biol 2013 Mar 7;33(6):1164-74. Epub 2013 Jan 7.

Department of Biochemistry and Molecular Genetics, University of Virginia, School of Medicine, Charlottesville, Virginia, USA.

RVB1/RVB2 (RuvBL1/RuvBL2 or pontin/reptin) are enigmatic AAA(+) ATPase proteins that are present in multiple cellular complexes. Although they have been implicated in many cellular functions, the exact molecular function of RVB proteins in the various complexes is not clear. TIP60 complex (TIP60.com) is a tumor suppressor chromatin-remodeling complex containing RVB proteins. RVBs are required for the lysine acetyltransferase activity of TIP60.com but not for that of the pure recombinant TIP60 polypeptide. Here we describe two molecular functions of RVBs in TIP60.com. First, RVBs negate the repression of catalytic activity of TIP60 by another protein in TIP60.com, p400. RVBs competitively displace the SNF2 domain of p400 from the TIP60 polypeptide. In addition RVBs are also required for heat stability of TIP60.com by a p400-independent pathway. RVB1 and RVB2 are redundant with each other for these functions and do not require their ATPase activities. Thus, RVB proteins act as molecular adaptors that can substitute for one another to facilitate the optimal assembly, heat stability, and function of the TIP60 complex.
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http://dx.doi.org/10.1128/MCB.01567-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3592018PMC
March 2013

Single-stranded DNA binding protein from human malarial parasite Plasmodium falciparum is encoded in the nucleus and targeted to the apicoplast.

Nucleic Acids Res 2010 Nov 22;38(20):7037-53. Epub 2010 Jun 22.

Special Centre for Molecular Medicine, School of Physical Sciences, Jawaharlal Nehru University, New Delhi 110067, India.

Apicoplast, an essential organelle of human malaria parasite Plasmodium falciparum contains a ∼35 kb circular genome and is a possible target for therapy. Proteins required for the replication and maintenance of the apicoplast DNA are not clearly known. Here we report the presence of single-stranded DNA binding protein (SSB) in P falciparum. PfSSB is targeted to the apicoplast and it binds to apicoplast DNA. A strong ssDNA binding activity specific to SSB was also detected in P. falciparum lysate. Both the recombinant and endogenous proteins form tetramers and the homology modelling shows the presence of an oligosaccharide/oligonucleotide-binding fold responsible for ssDNA binding. Additionally, we used SSB as a tool to track the mechanism of delayed death phenomena shown by apicoplast targeted drugs ciprofloxacin and tetracycline. We find that the transport of PfSSB is severely affected during the second life cycle following drug treatment. Moreover, the translation of PfSSB protein and not the transcription of PfSSB seem to be down-regulated specifically during second life cycle although there is no considerable change in protein expression profile between drug-treated and untreated parasites. These results suggest dual control of translocation and translation of apicoplast targeted proteins behind the delayed death phenomena.
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http://dx.doi.org/10.1093/nar/gkq565DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2978346PMC
November 2010

A unique 45-amino-acid region in the toprim domain of Plasmodium falciparum gyrase B is essential for its activity.

Eukaryot Cell 2009 Nov 21;8(11):1759-69. Epub 2009 Aug 21.

Special Centre for Molecular Medicine, Jawaharlal Nehru University, New Delhi 110067, India.

DNA gyrase is the only topoisomerase that can introduce negative supercoils into the DNA at the cost of ATP hydrolysis. Some but not all the steps of the topoisomerization reaction are understood clearly for both eukaryotic topoII and DNA gyrase. This study is an attempt to understand whether the B subunit of DNA gyrase binds to DNA directly, which may be central to the stimulation of its ATPase activity essential for gyrase function. We have dissected the Plasmodium falciparum gyrase B (PfGyrB) subunit to identify a 45-amino-acid region in the toprim domain that is responsible for its intrinsic DNA binding activity, DNA-stimulated ATPase activity, and DNA cleavage. We find that DNA has to enter through the ATP-operated clamp of PfGyrB to gain access to the DNA binding region. Furthermore, the rate of ATP hydrolysis of PfGyrB increases significantly with increasing DNA length, suggesting a possible communication between the ATPase domain and the DNA binding region that can account for its optimal ATPase activity. These results not only highlight the mechanism of GyrB action in the deadly human parasite P. falciparum but also provide meaningful insights into the current mechanistic model of DNA transport by gyrase during the topoisomerization reaction.
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http://dx.doi.org/10.1128/EC.00149-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2772398PMC
November 2009

Functional dissection of the catalytic carboxyl-terminal domain of origin recognition complex subunit 1 (PfORC1) of the human malaria parasite Plasmodium falciparum.

Eukaryot Cell 2009 Sep 24;8(9):1341-51. Epub 2009 Jul 24.

Special Centre for Molecular Medicine, Jawaharlal Nehru University, New Delhi, India.

Origin recognition complex subunit 1 (ORC1) is essential for DNA replication in eukaryotes. The deadly human malaria parasite Plasmodium falciparum contains an ORC1/CDC6 homolog with several interesting domains at the catalytic carboxyl-terminal region that include a putative nucleoside triphosphate-binding and hydrolysis domain, a putative PCNA-interacting-protein (PIP) motif, and an extreme C-terminal region that shows poor homology with other ORC1 homologs. Due to the unavailability of a dependable inducible gene expression system, it is difficult to study the structure and function of essential genes in Plasmodium. Using a genetic yeast complementation system and biochemical experiments, here we show that the putative PIP domain in ORC1 that facilitates in vitro physical interaction with PCNA is functional in both yeast (Saccharomyces cerevisiae) and Plasmodium in vivo, confirming its essential biological role in eukaryotes. Furthermore, despite having less sequence homology, the extreme C-terminal region can be swapped between S. cerevisiae and P. falciparum and it binds to DNA directly, suggesting a conserved role of this region in DNA replication. These results not only provide us a useful system to study the function of the essential genes in Plasmodium, they help us to identify the previously undiscovered unique features of replication proteins in general.
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http://dx.doi.org/10.1128/EC.00170-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2747822PMC
September 2009

Helicobacter pylori single-stranded DNA binding protein--functional characterization and modulation of H. pylori DnaB helicase activity.

FEBS J 2009 Jan 11;276(2):519-31. Epub 2008 Dec 11.

Special Centre for Molecular Medicine, Jawaharlal Nehru University, New Delhi, India.

Helicobacter pylori, an important bacterial pathogen, causes gastric ulcer and gastric adenocarcinoma in humans. The fundamentals of basic biology such as DNA replication are poorly understood in this pathogen. In the present study, we report the cloning and functional characterization of the single-stranded DNA (ssDNA) binding protein from H. pylori. The N-terminal DNA binding domain shows significant homology with E. coli single-stranded DNA binding protein (SSB), whereas the C-terminal domain shows less homology. The overall DNA-binding activity and tetramerization properties, however, remain unaffected. In in vitro experiments with purified proteins, H. pylori (Hp) SSB bound specifically to ssDNA and modulated the enzymatic ATPase and helicase activity of HpDnaB helicase. HpSSB and HpDnaB proteins were co-localized in sharp, distinct foci in exponentially growing H. pylori cells, whereas both were spread over large areas in its dormant coccoid form, suggesting the absence of active replication forks in the latter. These results confirm the multiple roles of SSB during DNA replication and provide evidence for altered replicative metabolism in the spiral and coccoid forms that may be central to the bacterial physiology and pathogenesis.
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http://dx.doi.org/10.1111/j.1742-4658.2008.06799.xDOI Listing
January 2009

The domain structure of Helicobacter pylori DnaB helicase: the N-terminal domain can be dispensable for helicase activity whereas the extreme C-terminal region is essential for its function.

Nucleic Acids Res 2007 11;35(9):2861-74. Epub 2007 Apr 11.

Special Centre for Molecular Medicine, Jawaharlal Nehru University, New Delhi 110067, India.

Hexameric DnaB type replicative helicases are essential for DNA strand unwinding along with the direction of replication fork movement. These helicases in general contain an amino terminal domain and a carboxy terminal domain separated by a linker region. Due to the lack of crystal structure of a full-length DnaB like helicase, the domain structure and function of these types of helicases are not clear. We have reported recently that Helicobacter pylori DnaB helicase is a replicative helicase in vitro and it can bypass Escherichia coli DnaC activity in vivo. Using biochemical, biophysical and genetic complementation assays, here we show that though the N-terminal region of HpDnaB is required for conformational changes between C6 and C3 rotational symmetry, it is not essential for in vitro helicase activity and in vivo function of the protein. Instead, an extreme carboxy terminal region and an adjacent unique 34 amino acid insertion region were found to be essential for HpDnaB activity suggesting that these regions are important for proper folding and oligomerization of this protein. These results confer great potential in understanding the domain structures of DnaB type helicases and their related function.
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http://dx.doi.org/10.1093/nar/gkm167DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1888833PMC
June 2007

Use of pedicled omentum in esophagogastric anastomosis for prevention of anastomotic leak.

Ann Thorac Surg 2006 Nov;82(5):1857-62

Department of Cardiovascular and Thoracic Surgery, Sher-i-Kashmir Institute of Medical Sciences, Srinagar, Kashmir, India.

Background: Esophagogastrectomy for carcinoma of the esophagus is the standard surgical treatment for cure or palliation. Esophagogastric anastomotic leakage is a life-threatening postoperative complication, more so if the leakage occurs in the chest.

Methods: A prospective, randomized study was conducted on 238 patients treated for carcinoma of the esophagus between January 2000 and January 2006. The study excluded 44 patients (18.49%) who were inoperable. The patients were assigned to two treatment groups of 97 each (A and B) according to a restricted permuted block randomization plan. Group A patients underwent esophagogastrectomy with wrapping of the pedicled omentum around the esophagogastric anastomosis. Group B patients underwent esophagogastrectomy without using the omental graft. An Ivor-Lewis type esophagogastrectomy (TTE) was done in 122 patients (62.89%) and a transhiatal esophagogastrectomy (THE) was done in 72 (37.11%).

Results: Anastomotic leaks occurred in 3 group A patients (3.09%) and in 14 (14.43%) group B patients. In group A, 54 patients underwent THE and 43 had TTE, with anastomotic leakage in 2 (3.70%) and 1 (2.33%) patients, respectively. In group B, 48 patients had THE and 49 had TTE, with anastomotic leakage in 8 (16.26%) and 6 (12.24%), respectively. The difference in the incidence of leakage was statistically significant (p = 0.005). There was no complication related to the omental graft technique nor was there a significant difference in the mortality between the two groups.

Conclusions: The pedicled omental transposition for reinforcing the anastomotic suture line significantly reduces the incidence of leakage after esophagogastrectomy for carcinoma of the esophagus, thus decreasing the morbidity and mortality of the procedure.
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http://dx.doi.org/10.1016/j.athoracsur.2006.05.101DOI Listing
November 2006
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