Plasmodium falciparum topoisomerases: Emerging targets for anti-malarial therapy.

Different metabolic pathways like DNA replication, transcription, and recombination generate topological constrains in the genome. These topological constraints are resolved by essential molecular machines known as topoisomerases. To bring changes in DNA topology, the topoisomerases create a single or double-stranded nick in the template DNA, hold the nicked ends to let the tangled DNA pass through, and finally re-ligate the breaks. The DNA nicking and re-ligation activities as well as ATPase activities (when present) in topoisomerases are subjected to inhibition by several anticancer and antibacterial drugs, thus establishing these enzymes as successful targets in anticancer and antibacterial therapies. The anti-topoisomerase drugs interfere with the functioning of these enzymes and result in the accumulation of DNA tangles or lethal genomic breaks, thereby promoting host cell (or organism) death. The potential of topoisomerases in the human malarial parasite, Plasmodium falciparum in antimalarial drug development has received little attention so far. Interestingly, the parasite genome encodes orthologs of topoisomerases found in eukaryotes, prokaryotes, and archaea, thus, providing an enormous opportunity for investigating these enzymes for antimalarial therapeutics. This review focuses on the features of Plasmodium falciparum topoisomerases (PfTopos) with respect to their closer counterparts in other organisms. We will discuss overall advances and basic challenges with topoisomerase research in Plasmodium falciparum and our attempts to understand the interaction of PfTopos with classical and new-generation topoisomerase inhibitors using in silico molecular docking approach. The recent episodes of parasite resistance against artemisinin, the only effective antimalarial drug at present, further highlight the significance of investigating new drug targets including topoisomerases in antimalarial therapeutics.

Flavonoids as promising molecules in the cancer therapy: An insight.

Cancer continues to increase global morbidity and mortality rates. Despite substantial progress in the development of various chemically synthesized anti-cancer drugs, the poor prognosis of the disease still remains a big challenge. The most common drawback of conventional cancer therapies is the emergence of drug resistance eventually leading to the discontinuation of chemotherapy. Moreover, advanced target-specific therapies including immunotherapy and stem cell therapy are expensive enough and are unaffordable for most patients in poorer nations. Therefore, alternative and cheaper therapeutic strategies are needed to complement the current cancer treatment approaches. Phytochemicals are bioactive compounds produced naturally by plants and have great potential in human health and disease. These compounds possess antiproliferative, anti-oxidant, and immunomodulatory properties. Among the phytochemicals, flavonoids are very effective in treating a wide range of diseases from cardiovascular diseases and immunological disorders to cancer. They scavenge reactive oxygen species (ROS), inhibit cancer metastasis, modulate the immune system and induce apoptotic or autophagic cell death in cancers. This review will discuss the potential of various phytochemicals particularly flavonoids in attempts to target various cancers.

Exploring the mTOR Signalling Pathway and Its Inhibitory Scope in Cancer.

Mechanistic target of rapamycin (mTOR) is a protein kinase that regulates cellular growth, development, survival, and metabolism through integration of diverse extracellular and intracellular stimuli. Additionally, mTOR is involved in interplay of signalling pathways that regulate apoptosis and autophagy. In cells, mTOR is assembled into two complexes, mTORC1 and mTORC2. While mTORC1 is regulated by energy consumption, protein intake, mechanical stimuli, and growth factors, mTORC2 is regulated by insulin-like growth factor-1 receptor (IGF-1R), and epidermal growth factor receptor (EGFR). mTOR signalling pathways are considered the hallmark in cancer due to their dysregulation in approximately 70% of cancers. Through downstream regulators, ribosomal protein S6 kinase ß-1 (S6K1) and eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1), mTORC1 influences various anabolic and catabolic processes in the cell. In recent years, several mTOR inhibitors have been developed with the aim of treating different cancers. In this review, we will explore the current developments in the mTOR signalling pathway and its importance for being targeted by various inhibitors in anti-cancer therapeutics.

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

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.

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

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 CRL4Cdt2 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.

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

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.

Potent antimalarial activity of acriflavine in vitro and in vivo.

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.

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

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.

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

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.

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

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.

RVBs are required for assembling a functional TIP60 complex.

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.

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

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.

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

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.

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

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.

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

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.

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.

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.