KREH1 RNA helicase activity promotes utilization of initiator gRNAs across multiple mRNAs in trypanosome RNA editing.

Mitochondrial U-indel RNA editing in kinetoplastid protozoa is directed by trans-acting gRNAs and mediated by a holoenzyme with associated factors. Here, we examine the function of the holoenzyme-associated KREH1 RNA helicase in U-indel editing. We show that KREH1 knockout (KO) impairs editing of a small subset of mRNAs. Overexpression of helicase-dead mutants results in expanded impairment of editing across multiple transcripts, suggesting the existence of enzymes that can compensate for KREH1 in KO cells. In depth analysis of editing defects using quantitative RT-PCR and high-throughput sequencing reveals compromised editing initiation and progression in both KREH1-KO and mutant-expressing cells. In addition, these cells exhibit a distinct defect in the earliest stages of editing in which the initiator gRNA is bypassed, and a small number of editing events takes place just outside this region. Wild type KREH1 and a helicase-dead KREH1 mutant interact similarly with RNA and holoenzyme, and overexpression of both similarly disorders holoenzyme homeostasis. Thus, our data support a model in which KREH1 RNA helicase activity facilitates remodeling of initiator gRNA-mRNA duplexes to permit accurate utilization of initiating gRNAs on multiple transcripts.

Selective nuclear export of mRNAs is promoted by DRBD18 in Trypanosoma brucei.

Kinetoplastids, including Trypanosoma brucei, control gene expression primarily at the posttranscriptional level. Nuclear mRNA export is an important, but understudied, step in this process. The general heterodimeric export factors, Mex67/Mtr2, function in the export of mRNAs and tRNAs in T. brucei, but RNA binding proteins (RBPs) that regulate export processes by controlling the dynamics of Mex67/Mtr2 ribonucleoprotein formation or transport have not been identified. Here, we report that DRBD18, an essential and abundant T. brucei RBP, associates with Mex67/Mtr2 in vivo, likely through its direct interaction with Mtr2. DRBD18 downregulation results in partial accumulation of poly(A)+ mRNA in the nucleus, but has no effect on the localization of intron-containing or mature tRNAs. Comprehensive analysis of transcriptomes from whole-cell and cytosol in DRBD18 knockdown parasites demonstrates that depletion of DRBD18 leads to impairment of nuclear export of a subset of mRNAs. CLIP experiments reveal the association of DRBD18 with several of these mRNAs. Moreover, DRBD18 knockdown leads to a partial accumulation of the Mex67/Mtr2 export receptors in the nucleus. Taken together, the current study supports a model in which DRBD18 regulates the selective nuclear export of mRNAs by promoting the mobilization of export competent mRNPs to the cytosol through the nuclear pore complex.

Trypanosoma brucei UMSBP2 is a single-stranded telomeric DNA binding protein essential for chromosome end protection.

Universal minicircle sequence binding proteins (UMSBPs) are CCHC-type zinc-finger proteins that bind a single-stranded G-rich sequence, UMS, conserved at the replication origins of the mitochondrial (kinetoplast) DNA of trypanosomatids. Here, we report that Trypanosoma brucei TbUMSBP2, which has been previously proposed to function in the replication and segregation of the mitochondrial DNA, colocalizes with telomeres at the nucleus and is essential for their structure, protection and function. Knockdown of TbUMSBP2 resulted in telomere clustering in one or few foci, phosphorylation of histone H2A at the vicinity of the telomeres, impaired nuclear division, endoreduplication and cell growth arrest. Furthermore, TbUMSBP2 depletion caused rapid reduction in the G-rich telomeric overhang, and an increase in C-rich single-stranded telomeric DNA and in extrachromosomal telomeric circles. These results indicate that TbUMSBP2 is essential for the integrity and function of telomeres. The sequence similarity between the mitochondrial UMS and the telomeric overhang and the finding that UMSBPs bind both sequences suggest a common origin and/or function of these interactions in the replication and maintenance of the genomes in the two organelles. This feature could have converged or preserved during the evolution of the nuclear and mitochondrial genomes from their ancestral (likely circular) genome in early diverged protists.

Isobenzofuranone derivatives exhibit antileishmanial effect by inhibiting type II DNA topoisomerase and inducing host response.

Leishmania, a protozoan parasite, causes a wide range of human diseases ranging from the localized self-healing cutaneous lesions to fatal visceral leishmaniasis. Toxicity of traditional first line drugs and emergence of drug-resistant strains have worsened the situation. DNA topoisomerase II in kinetoplastid protozoan parasites are of immense interest as drug target because they take part in replication of unusual kinetoplast DNA network. In this study, we have taken target-based therapeutic approaches to combat leishmaniasis. Two isobenzofuranone compounds, viz., (1) 3,5-bis(4-chlorophenyl)-7-hydroxyisobenzofuran-1(3H)-one (JVPH3) and (2) (4-bromo)-3'-hydroxy-5'-(4-bromophenyl)-benzophenone(JVPH4) were synthesized chemically and characterized by NMR and mass spectrometry analysis. Activity of type II DNA topoisomerase of leishmania (LdTOPII) was monitored by decatenation assay and plasmid cleavage assay. The antiparasitic activity of these compounds was checked in experimental BALB/c mice model of visceral leishmaniasis. Isobenzofuranone derivatives exhibited potent antileishmanial effect on both antimony (Sb) sensitive and resistant parasites. Treatment with isobenzofuranone derivatives on promastigotes caused induction of reactive oxygen species (ROS)-mediated apoptosis like cell death in leishmania. Both the compounds inhibited the decatenation activity of LdTOPII but have no effect on bi-subunit topoisomerase IB. Treatment of LdTOPII with isobenzofuranone derivatives did not stabilize cleavage complex formation both in vitro and in vivo. Moreover, treatment with isobenzofuranone derivatives on Leishmania donovani-infected mice resulted in clearance of parasites in liver and spleen by induction of Th1 cytokines. Taken together, our data suggest that these compounds can be exploited as potential antileishmanial agents targeted to DNA topoisomerase II of the parasite.

Flavone-resistant Leishmania donovani overexpresses LdMRP2 transporter in the parasite and activates host MRP2 on macrophages to circumvent the flavone-mediated cell death.

In parasites, ATP-binding cassette (ABC) transporters represent an important family of proteins related to drug resistance and other biological activities. Resistance of leishmanial parasites to therapeutic drugs continues to escalate in developing countries, and in many instances, it is due to overexpressed ABC efflux pumps. Progressively adapted baicalein (BLN)-resistant parasites (pB(25)R) show overexpression of a novel ABC transporter, which was classified as ABCC2 or Leishmania donovani multidrug resistance protein 2 (LdMRP2). The protein is primarily localized in the flagellar pocket region and in internal vesicles. Overexpressed LdABCC2 confers substantial BLN resistance to the parasites by rapid drug efflux. The BLN-resistant promastigotes when transformed into amastigotes in macrophage cells cannot be cured by treatment of macrophages with BLN. Amastigote resistance is concomitant with the overexpression of macrophage MRP2 transporter. Reporter analysis and site-directed mutagenesis assays demonstrated that antioxidant response element 1 is activated upon infection. The expression of this phase II detoxifying gene is regulated by NFE2-related factor 2 (Nrf2)-mediated antioxidant response element activation. In view of the fact that the signaling pathway of phosphoinositol 3-kinase controls microfilament rearrangement and translocation of actin-associated proteins, the current study correlates with the intricate pathway of phosphoinositol 3-kinase-mediated nuclear translocation of Nrf2, which activates MRP2 expression in macrophages upon infection by the parasites. In contrast, phalloidin, an agent that prevents depolymerization of actin filaments, inhibits Nrf2 translocation and Mrp2 gene activation by pB(25)R infection. Taken together, these results provide insight into the mechanisms by which resistant clinical isolates of L. donovani induce intracellular events relevant to drug resistance.

The lignan glycosides lyoniside and saracoside poison the unusual type IB topoisomerase of Leishmania donovani and kill the parasite both in vitro and in vivo.

Lignans are diphenyl propanoids with vast range of biological activities. The present study provides an important insight into the anti-leishmanial activities of two lignan glycosides, viz. lyoniside and saracoside. These compounds inhibit catalytic activities of topoisomerase IB (LdTopIB) of Leishmania donovani in non-competitive manner and stabilize the LdTopIB mediated cleavage complex formation both in vitro and in Leishmania promastigotes and subsequently inhibit the religation of cleaved strand. These two compounds not only poison LdTopIB but also can interact with the free enzyme LdTopIB. We have also shown that lyoniside and saracoside are cytotoxic to promastigotes and intracellular amastigotes. The protein-DNA complex formation leads to double strand breaks in DNA which ultimately triggers apoptosis-like cell death in the parasite. Along with their cytotoxicity towards sodium antimony gluconate (SAG) sensitive AG83 strain, their ability to kill SAG resistant GE1 strain makes these two compounds potential anti-leishmanial candidates. Not only they effectively kill L. donovani amastigotes inside macrophages in vitro, lyoniside and saracoside demonstrated strong anti-leishmanial efficacies in BALB/c mice model of leishmaniasis. Treatment with these lignan glycosides produce nitric oxide and reactive oxygen species which result in almost complete clearance of the liver and splenic parasite burden. These compounds do not inhibit human topoisomerase IB upto 200µM concentrations and had poor cytotoxic effect on uninfected cultured murine peritoneal macrophages upto 100µM concentrations. Taken together it can be concluded that these compounds can be developed into excellent therapeutic agent against deadly disease leishmaniasis.

The gelatinases and their inhibitors: the structure-activity relationships.

The interest in gelatinases is increased because of their association in diverse human diseases, though the relationship between MMP expression and disease progression is very complex and varies in cell to cell. Targeting gelatinases in disease treatment is complicated by the fact that gelatinases are indispensable for normal development and physiology due to their multifunctionality, possible functional redundancy, context-dependent expression, and activity. They are secreted as inactive zymogens which are processed to become active by removal of N-terminal propeptide. The folded conformation of zymogen is required to keep the gelatinases in its latency. Acting on a broad spectrum of extracellular substrates, the gelatinases (both MMP-2 and MMP-9) are critical to the biological processes. Three-dimensional structures of gelatinase-inhibitor complexes and inhibition profiles of compounds screened on them provide an invaluable source to gain insight into the structural determinants as well as functional selectivity. The quest for selective MMP inhibitors (MMPIs) still remains a challenge in search of successful clinical candidates. An increased understanding of the structure, regulation, and function of the individual MMPs will likely lead to more effective strategies in the development of highly selective inhibitors for any given MMP that can then be exploited to achieve the desired drugs.

An alternative therapy for idiopathic pulmonary fibrosis by doxycycline through matrix metalloproteinase inhibition.

BACKGROUND: Idiopatiic pulmonary fibrosis (IPF) is a disease of dysregulated fibrogenesis with abnormal matrix metalloproteinase (MMPs) activity, angiogenesis, and profibrotic milieu wherein MMPs inhibition appears to be target-based therapy. We evaluated the role of doxycycline as a nonspecific inhibitor of MMPs in IPF patients. MATERIALS AND METHODS: Patients of IPF diagnosed on the basis of ATS-ERS consensus criteria were put on oral doxycycline in an open prospective trial. They were followed up for long term with spirometry, 6 min walk test (6MWT), St. Georges respiratory questionnaire (SGRQ), forced vital capacity (FVC), and repeat bronchoscopy while on doxycycline monotherapy for over 24 weeks. Both the initial and follow-up broncho alveolar lavage fluids (BALF) from IPF patients (n = 6) and control subjects (n = 6) were looked for MMP-9, -3, tissue inhibitor of metalloproteinase (TIMP)-1 and vascular endothelial growth factor (VEGF) expression. Additionally, doxycycline's action on MMP activities in vitro was tested in BALF of IPF patients. RESULTS: Doxycycline intervention showed significant improvement in IPF patients in terms of change in 6MWT, SGRQ, FVC, and quality of life. The level of MMP-9, -3, TIMP-1 and VEGF in the BALF were found significantly higher in the IPF patients compared to the controls while doxycycline therapy reduced those parameters nearer to control value. Doxycycline also showed a significant dose-dependent reduction in the in vitro MMPs activities in BALF. CONCLUSION: Doxycycline shows significant prospect in the treatment of IPF through its anti MMPs activities. This is the first report on a case series of long-term doxycycline monotherapy in IPF patients.

Doxycycline blocks gastric ulcer by regulating matrix metalloproteinase-2 activity and oxidative stress.

AIM: To examine the effect of doxycycline on the activity of matrix metalloproteinases (MMPs) and oxidative stress in gastric tissues of rats following gastric injury. METHODS: Gastric ulcers were generated in rats by administration of 70% ethanol, and activity of doxycycline was tested by administration 30 min prior to ethanol. Similarly, the effect of doxycycline was tested in an indomethacin-induced gastric ulcer model. The activities and expression of MMPs were examined by zymography and Western blot analysis. RESULTS: Gastric injury in rats as judged by elevated ulcer indices following exposure to ulcerogen, either indomethacin or ethanol, was reversed significantly by doxycycline. Indomethacin-induced ulcerated gastric tissues exhibited about 12-fold higher proMMP-9 activity and about 5-fold higher proMMP-3 activity as compared to control tissues. Similarly, ethanol induced about 22-fold and about 6-fold higher proMMP-9 and proMMP-3 activities, respectively, in rat gastric tissues. Both proMMP-9 and MMP-3 activities were markedly decreased by doxycycline in ulcerogen treated rat gastric tissues. In contrast, the reduced MMP-2 activity in ulcerated tissues was increased by doxycycline during ulcer prevention. On the other hand, doxycycline inhibited significantly proMMP-9, -2 and -3 activities in vitro. In addition, doxycycline reduced oxidative load in gastric tissues and scavenged H2O2 in vitro. Our results suggest a novel regulatory role of doxycycline on MMP-2 activity in addition to inhibitory action on MMP-9 and MMP-3 during prevention of gastric ulcers. CONCLUSION: This is the first demonstration of dual action of doxycycline, that is, regulation of MMP activity and reduction of oxidative stress in arresting gastric injury.

Downregulation of matrix metalloproteinase-9 by melatonin during prevention of alcohol-induced liver injury in mice.

Matrix metalloproteinases (MMPs) have been implicated in inflammatory and degradative processes in several diseases. The study aims to explore the mechanism of MMP-9 regulation in alcohol-induced acute liver injury and its protection by melatonin in mice. Alcohol-induced acute liver injury was induced in female Balb/C mice by ethanol administration and protection studies were carried out with a well-known antioxidant molecule, melatonin. Degree of liver injury was monitored by histological and biochemical analysis of liver tissues. Oral administration of ethanol in mouse caused significant increase in alanine amino transferase (ALT) activity in serum. Depletion of glutathione and enhancement of lipid peroxidation as well as protein oxidation was observed in liver tissues following ethanol treatment. However, melatonin exhibited potent hepatoprotective activity by inhibiting ALT activity and oxidative stress. Additionally, MMP-9 expression was increased by ethanol in a dose and time dependent manner in liver tissue and serum. Increased secretion of proMMP-9 was strongly correlated with the expression of proinflammatory cytokines e.g., tumor necrosis factor (TNF)-α, interleukin (IL)-1ß and IL6. Melatonin showed hepatoprotective role by downregulation of MMP-9 and upregulation of tissue inhibitor of metalloproteases (TIMP-1) expression in liver tissue. Nuclear factor (NF)-κB, plays an important role in inducing inflammatory genes during oxidative stress, thus the role of NF-κB in ethanol-induced liver injury was investigated. Ethanol induced nuclear translocation of NF-κB and increased degradation of inhibitor of NF-κB (IκBα) in liver tissues. Moreover, ethanol-induced NF-κB translocation into nucleus was inhibited significantly by melatonin. This is the first study to elucidate the induction of MMP-9 expression by NF-κB-dependent pathway in ethanol-induced acute liver injury in mice. This study also identifies the novel role of melatonin in hepatoprotection via MMP-9 down regulation.

Novel role of famotidine in downregulation of matrix metalloproteinase-9 during protection of ethanol-induced acute gastric ulcer.

Gastric ulcer is a multifaceted process including acid secretion, reactive oxygen species generation, prostaglandin inhibition, and extracellular matrix (ECM) degradation. Matrix metalloproteinases (MMPs) have the ability to cleave and remodel the ECM. We investigated the activity and expression of MMP-9 and -2 in ethanol-induced acute gastric ulceration in rats. We found that severity of gastric ulcer was strongly correlated with increasing doses of ethanol and increased secretion of proMMP-9. ProMMP-9 was upregulated approximately 25-fold at maximum ulcer index. Increased secretion of proMMP-9 was associated with increased expression of tumor necrosis factor-alpha and interleukin-6. We examined the effect of H(2)-receptor antagonists and antioxidants on proMMP-9 secretion and synthesis during prevention of ethanol-induced gastric ulcer. Our data reveal that famotidine dose dependently blocked increased secretion and synthesis of proMMP-9 during gastroprotection and arrested infiltration of inflammatory cells as well as oxidative stress in rat gastric tissues. Similar to H(2)-receptor antagonists, N-acetylcysteine and dimethyl sulfoxide, well-known antioxidants, inhibited proMMP-9 upregulation to the control level. In conclusion, ethanol-induced gastric ulceration is associated with increased expression of proMMP-9 that can be attenuated by H(2)-receptor antagonists and antioxidants. These findings furnish a novel MMP-9-mediated pathway and its inhibition via proinflammatory cytokines by famotidine in ethanol-induced gastric ulceration.

Matrix metalloproteinase-9 activity and expression is reduced by melatonin during prevention of ethanol-induced gastric ulcer in mice.

Matrix metalloproteinases (MMPs) play an important role in degradation of gastric extracellular matrix proteins. However, no reports are available on the relationship between the activity of MMPs and gastric ulceration induced by alcohol. Our objective was to investigate the effect of melatonin (N-acetyl-5-methoxytryptamine) on the regulation of MMP-9 and MMP-2 activities during prevention of ethanol-induced gastric ulcer. Biochemical and zymographic methods were used to analyze MMP-9 and -2 activities in gastric tissues of Balb/c mice following induction of gastric ulcer by ethanol. Our studies reveal that melatonin arrested cell injury, protein carbonyl formation, and lipid peroxidation in mice during gastroprotection. Melatonin dose-dependently reduced proMMP-9 activity that was induced ( approximately 25-fold) during ethanol-induced gastric ulceration. Severity of gastric ulcers were correlated proportionately with increased dose of ethanol and elevated activity of proMMP-9 and -2. The reduced activities of MMP-9 and -2 were associated with reduced expression of TNF-alpha and increased expression of tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2). We conclude that melatonin's ability to prevent ethanol-induced gastric ulceration in mice is related to a reduction in proMMP-9 activity and expression.