Heat Shock Protein 90 as a Drug Target against Protozoan Infections: BIOCHEMICAL CHARACTERIZATION OF HSP90 FROM PLASMODIUM FALCIPARUM AND TRYPANOSOMA EVANSI AND EVALUATION OF ITS INHIBITOR AS A CANDIDATE DRUG*

Using a pharmacological inhibitor of Hsp90 in cultured malarial parasite, we have previously implicated Plasmodium falciparum Hsp90 (PfHsp90) as a drug target against malaria. In this study, we have biochemically characterized PfHsp90 in terms of its ATPase activity and interaction with its inhibitor geldanamycin (GA) and evaluated its potential as a drug target in a preclinical mouse model of malaria. In addition, we have explored the potential of Hsp90 inhibitors as drugs for the treatment of Trypanosoma infection in animals. Our studies with full-length PfHsp90 showed it to have the highest ATPase activity of all known Hsp90s; its ATPase activity was 6 times higher than that of human Hsp90. Also, GA brought about more robust inhibition of PfHsp90 ATPase activity as compared with human Hsp90. Mass spectrometric analysis of PfHsp90 expressed in P. falciparum identified a site of acetylation that overlapped with Aha1 and p23 binding domain, suggesting its role in modulating Hsp90 multichaperone complex assembly. Indeed, treatment of P. falciparum cultures with a histone deacetylase inhibitor resulted in a partial dissociation of PfHsp90 complex. Furthermore, we found a well known, semisynthetic Hsp90 inhibitor, namely 17-(allylamino)-17-demethoxygeldanamycin, to be effective in attenuating parasite growth and prolonging survival in a mouse model of malaria. We also characterized GA binding to Hsp90 from another protozoan parasite, namely Trypanosoma evansi. We found 17-(allylamino)-17-demethoxygeldanamycin to potently inhibit T. evansi growth in a mouse model of trypanosomiasis. In all, our biochemical characterization, drug interaction, and animal studies supported Hsp90 as a drug target and its inhibitor as a potential drug against protozoan diseases.     

Carbonic anhydrase inhibitors. Inhibition of mammalian isoforms I–XIV with a series of natural product polyphenols and phenolic acids

A series of phenolic acids and phenol natural products, such as p-hydroxybenzoic acid, p-coumaric acid, caffeic acid, ferulic acid, gallic acid, syringic acid, quercetin, and ellagic acid, were investigated for their inhibitory effects against the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). All mammalian isozymes of human (h) or murine (m) origin hCA I–hCA XII, mCA XIII and hCA XIV were inhibited in the low micromolar or submicromolar range by these (poly)phenols (K_Is in the range of 0.87–7.79 μM). p-Hydroxybenzoic acid was the best inhibitor of all isozymes (K_Is of 0.87–35.4 μM) and the different isozymes showed very variable inhibition profiles with these derivatives. Phenols like the ones investigated here possess a CA inhibition mechanism distinct of that of the sulfonamides/sulfamates used clinically or the coumarins. Unlike the sulfonamides, which bind to the catalytic zinc ion, phenols are anchored at the Zn(II)-coordinated water molecule and bind more externally within the active site cavity, making contacts with various amino acid residues. As this is the region with the highest variability between the many CA isozymes found in mammals, this class of compounds may lead to isoform-selective inhibitors targeting just one or few of the medicinally relevant CAs.     

Revealing on hydrogen sulfide and nitric oxide signals co-ordination for plant growth under stress conditions

In the recent times, plants are facing certain types of environmental stresses, which give rise to formation of reactive oxygen species (ROS) such as hydroxyl radicals, hydrogen peroxides, superoxide anions and so on. These are required by the plants at low concentrations for signal transduction and at high concentrations, they repress plant root growth. Apart from the ROS activities, hydrogen sulfide (H₂S) and nitric oxide (NO) have major contributions in regulating growth and developmental processes in plants, as they also play key roles as signaling molecules and act as chief plant immune defense mechanisms against various biotic as well as abiotic stresses. H₂S and NO are the two pivotal gaseous messengers involved in growth, germination and improved tolerance in plants under stressed and non-stress conditions. H₂S and NO mediate cell signaling in plants as a response to several abiotic stresses like temperature, heavy metal exposure, water and salinity. They alter gene expression levels to induce the synthesis of antioxidant enzymes, osmolytes and also trigger their interactions with each other. However, research has been limited to only cross adaptations and signal transductions. Understanding the change and mechanism of H₂S and NO mediated cell signaling will broaden our knowledge on the various biochemical changes that occur in plant cells related to different stresses. A clear understanding of these molecules in various environmental stresses would help to confer biotechnological applications to protect plants against abiotic stresses and to improve crop productivity.     

Identification and Molecular Characterization of a Novel Type of α-galactosidase from Pyrococcus furiosus

An α-galactosidase gene from Pyrococcus furiosus was identified, cloned and functionally expressed in Escherichia coli. It is the first α-galactosidase from a hyperthermophilic archaeon described to date. The gene encodes a unique amino acid sequence compared to other α-galactosidases. Highest homology was found with α-amylases classified in family 57 of glycoside hydrolases. The 364 amino acid protein had a calculated mass of 41.6 kDa. The recombinant α-galactosidase specifically catalyzed the hydrolysis of para-nitrophenyl-α-galactopyranoside, and to some extent that of melibiose and raffinose. The enzyme proved to be an extremely thermo-active and thermostable α-galactosidase with a temperature optimum of 115°C and a half-life time of 15 hours at 100°C. The pH optimum is between 5.0 and 5.5. Sequence analysis showed four conserved carboxylic residues. Site-directed mutagenesis was applied to identify the potential catalytic residues. Glu117Ala showed decreased enzyme activity, which could be rescued by the addition of azide or formate. It is concluded that glutamate 117 is the catalytic nucleophile, whereas the acid/base catalyst remains to be identified.     

HIP/PAP, a C-type lectin overexpressed in hepatocellular carcinoma, binds the RII alpha regulatory subunit of cAMP-dependent protein kinase and alters the cAMP-dependent protein kinase signalling.

HIP/PAP is a C-type lectin overexpressed in hepatocellular carcinoma (HCC). Pleiotropic biological activities have been ascribed to this protein, but little is known about the function of HIP/PAP in the liver. In this study, therefore, we searched for proteins interacting with HIP/PAP by screening a HCC cDNA expression library. We have identified the RII alpha regulatory subunit of cAMP-dependent protein kinase (PKA) as a partner of HIP/PAP. HIP/PAP and RII alpha were coimmunoprecipitated in HIP/PAP expressing cells. The biological relevance of the interaction between these proteins was established by demonstrating, using fractionation methods, that they are located in a same subcellular compartment. Indeed, though HIP/PAP is a protein secreted via the Golgi apparatus we showed that a fraction of HIP/PAP escaped the secretory apparatus and was recovered in the cytosol. Basal PKA activity was increased in HIP/PAP expressing cells, suggesting that HIP/PAP may alter PKA signalling. Indeed, we showed, using a thymidine kinase-luciferase reporter plasmid in which a cAMP responsive element was inserted upstream of the thymidine kinase promoter, that luciferase activity was enhanced in HIP/PAP expressing cells. Thus our findings suggest a novel mechanism for the biological activity of the HIP/PAP lectin.     

sickle, a novel Drosophila death gene in the reaper/hid/grim region, encodes an IAP-inhibitory protein

Inhibitors of apoptosis proteins (IAPs) interact with caspases and inhibit their protease activity, whereas the IAP-inhibitory proteins Smac/DIABLO in mammals and Reaper, Hid, and Grim in flies relieve IAP-mediated inhibition to induce cell death. Here we describe the functional characterization of the novel Drosophila cell death protein Sickle (Skl), which binds to IAPs and neutralizes their apoptotic inhibitory activity. Skl exhibits no sequence homology to Reaper, Hid, Grim, or Smac/DIABLO, except within the 4 residue N-terminal IAP binding motif. Skl interacts with Drosophila and mammalian IAPs and can promote caspase activation in the presence of IAPs. Consistent with these findings, expression of Skl in Drosophila and mammalian cell lines or in Drosophila embryos induces apoptosis. Skl can also synergize with Grim to induce cell death in the Drosophila eye imaginal disc. Based on biochemical and structural data, the N terminus of Skl, like that of the mammalian Smac/DIABLO, is absolutely required for its apoptotic and caspase-promoting activities and its ability to interact with IAPs. These findings point to conservation in the structure and function of the IAP-inhibitory proteins across species and suggest the existence of other family members.     

Natural killer cell lytic granule secretion occurs through a pervasive actin network at the immune synapse

Accumulation of filamentous actin (F-actin) at the immunological synapse (IS) is a prerequisite for the cytotoxic function of natural killer (NK) cells. Subsequent to reorganization of the actin network, lytic granules polarize to the IS where their contents are secreted directly toward a target cell, providing critical access to host defense. There has been limited investigation into the relationship between the actin network and degranulation. Thus, we have evaluated the actin network and secretion using microscopy techniques that provide unprecedented resolution and/or functional insight. We show that the actin network extends throughout the IS and that degranulation occurs in areas where there is actin, albeit in sub-micron relatively hypodense regions. Therefore we propose that granules reach the plasma membrane in clearances in the network that are appropriately sized to minimally accommodate a granule and allow it to interact with the filaments. Our data support a model whereby lytic granules and the actin network are intimately associated during the secretion process and broadly suggest a mechanism for the secretion of large organelles in the context of a cortical actin barrier.