Crystallization and preliminary X-ray studies of the C-terminal domain of Mycobacterium tuberculosis LexA

The C-terminal domain of Mycobacterium tuberculosis LexA has been crystallized in two different forms. The form 1 and form 2 crystals belonged to space groups P3(1)21 and P3(1), respectively. Form 1 contains one domain in the asymmetric unit, while form 2 contains six crystallographically independent domains. The structures have been solved by molecular replacement.

Stabilization of the bio-membrane by small molecules: interaction of trehalose with the phospholipid bilayer

Anhydrobiotic organisms undergo periods of acute dehydration during their life cycle. It is of interest to understand how the biomembrane remains intact through such stress. A disaccharide, trehalose, which is metabolised during anhydrobiosis is found to prevent disruption of model membrane systems. Molecular modelling techniques are used to investigate the possible mode of interaction of trehalose with a model monolayer. The objective is to maximise hydrogen bonding between the two systems. A phospholipid matrix consisting of 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) is chosen to represent the monolayer. The crystal structure of DMPC reveals that there are two distinct conformers designated as A and B. An expansion of the monolayer, coplanar with its surface, results in the trehalose molecule being accommodated in a pocket formed by four B conformers. One glucose ring of the sugar rests on the hydrophobic patch provided by the choline methyls of an A conformer. Five hydrogen bonds are formed involving the phosphate oxygens of three of the surrounding B conformers. The model will be discussed with reference to relevant experimental data on the interaction.

Innate Immune Recognition of RNA-virus infection in human macrophages

Innate immunity and host defence are rapidly evoked by structurally invariant molecular motifs common to microbial world, called pathogen associated molecular patterns (PAMPs). In addition to PAMPs, endogenous molecules released in response to inflammation and tissue damage, danger associated molecular patterns (DAMPs), are required for eliciting the response. The most important PAMPs of viruses are viral nucleic acids, their genome or its replication intermediates, whereas the identity and characteristics of virus infection-induced DAMPs are poorly defined. PAMPs and DAMPs engage a limited set of germ-line encoded pattern recognition receptors (PRRs) in immune and non-immune cells. Membrane-bound Toll-like receptors (TLRs), cytoplasmic retinoic acid inducible gene-I (RIG-I)-like receptors (RLRs) and nucleotide-binding oligomerization domain-like receptor (NLRs) are important PRRs involved in the recognition of the molecular signatures of viral infection, such as double-stranded ribonucleic acids (dsRNAs). Engagement of PRRs results in local and systemic innate immune responses which, when activated against viruses, evoke secretion of antiviral and pro-inflammatory cytokines, and programmed cell death i.e., apoptosis of the virus-infected cell. Macrophages are the central effector cells of innate immunity. They produce significant amounts of antiviral cytokines, called interferons (IFNs), and pro-inflammatory cytokines, such as interleukin (IL)-1β and IL-18. IL-1β and IL-18 are synthesized as inactive precursors, pro-IL-1β and pro-IL-18, that are processed by caspase-1 in a cytoplasmic multiprotein complex, called the inflammasome. After processing, these cytokines are biologically active and will be secreted. The signals and secretory routes that activate inflammasomes and the secretion of IL-1β and IL-18 during virus infections are poorly characterized. The main goal of this thesis was to characterize influenza A virus-induced innate immune responses and host-virus interactions in human primary macrophages during an infection. Methodologically, various techniques of cellular and molecular biology, as well as proteomic tools combined with bioinformatics, were utilized. Overall, the thesis provides interesting insights into inflammatory and antiviral innate immune responses, and has characterized host-virus interactions during influenza A virus-infection in human primary macrophages.

Hantavirus glycoprotein GN in virion assembly and regulation of interferon response

Hantaviruses have a tri-segmented negative-stranded RNA genome. The S segment encodes the nucleocapsid protein (N), M segment two glycoproteins, Gn and Gc, and the L segment the RNA polymerase. Gn and Gc are co-translationally cleaved from a precursor and targeted to the cis-Golgi compartment. The Gn glycoprotein consists of an external domain, a transmembrane domain and a C-terminal cytoplasmic domain. In addition, the S segment of some hantaviruses, including Tula and Puumala virus, have an open reading frame (ORF) encoding a nonstructural potein NSs that can function as a weak interferon antagonist. The mechanisms of hantavirus-induced pathogenesis are not fully understood but it is known that both hemorrhagic fever with renal syndrome (HFRS) and hantavirus (cardio) pulmonary syndrome (HCPS) share various features such as increased capillary permeability, thrombocytopenia and upregulation of TNF-. Several hantaviruses have been reported to induce programmed cell death (apoptosis), such as TULV-infected Vero E6 cells which is known to be defective in interferon signaling. Recently reports describing properties of the hantavirus Gn cytoplasmic tail (Gn-CT) have appeared. The Gn-CT of hantaviruses contains animmunoreceptor tyrosine-based activation motif (ITAM) which directs receptor signaling in immune and endothelial cells; and contain highly conserved classical zinc finger domains which may have a role in the interaction with N protein. More functions of Gn protein have been discovered, but much still remains unknown. Our aim was to study the functions of Gn protein from several aspects: synthesis, degradation and interaction with N protein. Gn protein was reported to inhibit interferon induction and amplication. For this reason, we also carried out projects studying the mechanisms of IFN induction and evasion by hantavirus. We first showed degradation and aggresome formation of the Gn-CT of the apathogenic TULV. It was reported earlier that the degradation of Gn-CT is related to the pathogenicity of hantavirus. We found that the Gn-CT of the apathogenic hantaviruses (TULV, Prospect Hill virus) was degraded through the ubiquitin-proteasome pathway, and TULV Gn-CT formed aggresomes upon treatment with proteasomal inhibitor. Thus the results suggest that degradation and aggregation of the Gn-CT may be a general property of most hantaviruses, unrelated to pathogenicity. Second, we investigated the interaction of TULV N protein and the TULV Gn-CT. The Gn protein is located on the Golgi membrane and its interaction with N protein has been thought to determine the cargo of the hantaviral ribonucleoprotein which is an important step in virus assembly, but direct evidence has not been reported. We found that TULV Gn-CT fused with GST tag expressed in bacteria can pull-down the N protein expressed in mammalian cells; a mutagenesis assay was carried out, in which we found that the zinc finger motif in Gn-CT and RNA-binding motif in N protein are indispensable for the interaction. For the study of mechanisms of IFN induction and evasion by Old World hantavirus, we found that Old World hantaviruses do not produce detectable amounts of dsRNA in infected cells and the 5 -termini of their genomic RNAs are monophosphorylated. DsRNA and tri-phosphorylated RNA are considered to be critical activators of innate immnity response by interacting with PRRs (pattern recognition receptors). We examined systematically the 5´-termini of hantavirus genomic RNAs and the dsRNA production by different species of hantaviruses. We found that no detectable dsRNA was produced in cells infected by the two groups of the old world hantaviruses: Seoul, Dobrava, Saaremaa, Puumala and Tula. We also found that the genomic RNAs of these Old World hantaviruses carry 5´-monophosphate and are unable to trigger interferon induction. The antiviral response is mainly mediated by alpha/beta interferon. Recently the glycoproteins of the pathogenic hantaviruses Sin Nombre and New York-1 viruses were reported to regulate cellular interferon. We found that Gn-CT can inhibit the induction of IFN activation through Toll-like receptor (TLR) and retinoic acid-inducible gene I-like RNA helicases (RLH) pathway and that the inhibition target lies at the level of TANK-binding kinase 1 (TBK-1)/ IKK epislon complex and myeloid differentiation primary response gene (88) (MyD88) / interferon regulatory factor 7 (IRF-7) complex.

Crystal Structure of Staphylococcus aureus Metallopeptidase (Sapep) Reveals Large Domain Motions between the Manganese-bound and Apo-states

Proteases belonging to the M20 family are characterized by diverse substrate specificity and participate in several metabolic pathways. The Staphylococcus aureus metallopeptidase, Sapep, is a member of the aminoacylase-I/M20 protein family. This protein is a Mn2+-dependent dipeptidase. The crystal structure of this protein in the Mn2+-bound form and in the open, metal-free state suggests that large interdomain movements could potentially regulate the activity of this enzyme. We note that the extended inactive conformation is stabilized by a disulfide bond in the vicinity of the active site. Although these cysteines, Cys(155) and Cys(178), are not active site residues, the reduced form of this enzyme is substantially more active as a dipeptidase. These findings acquire further relevance given a recent observation that this enzyme is only active in methicillin-resistant S. aureus. The structural and biochemical features of this enzyme provide a template for the design of novel methicillin-resistant S. aureus-specific therapeutics.

Mode of action of antitumour antibiotics-III: Modulation of permeability of nuclear membrane in the presence of the antibiotics

The binding characteristics of the antibiotics to nuclei and their effect on the permeability of nuclear membrane with respect to histones and ribonucleic acids have been investigated. The binding constant for chromomycin A3 was found to be 1.4 × 104M?1 and number of binding sites was equal to 3.48 ± 1.08 × 1012 molecules/nuclei. The antibiotic chromomycin A3 enhanced the uptake of lysine-rich histone, actinomycin D decreased the uptake and ethidium bromide had no effect. Chromomycin A3 also enhanced the release of acid insoluble fraction containing RNA from the nuclei, actinomycin D and ethidium bromide inhibited the release of acid insoluble fraction containing RNA. The relevance of this finding to the role of nuclear envelope in understanding the mechanism of action of the antibiotic has been discussed.

Terahertz Time Domain Spectroscopy to Detect Low-Frequency Vibrations of Double-Walled Carbon Nanotubes

We have measured the frequency-dependent real index of refraction and extinction coefficient (and hence the complex dielectric function) of a free-standing double-walled carbon nanotube film of thickness 200 nm by using terahertz time domain spectroscopy in the frequency range 0.1 to 2.5 THz. The real index of refraction and extinction coefficient have very high values of approximately 52 and 35, respectively, at 0.1 THz, which decrease at higher frequencies. Two low-frequency phonon modes of the carbon nanotubes at 0.45 and 0.75 THz were clearly observed for the first time in the real and imaginary parts of the complex dielectric function along with a broad resonance centred at around 1.45 THz, the latter being similar to that in single-walled carbon nanotubes assigned to electronic excitations. Our experiments bring out a possible application of double-walled carbon nanotube films as a neutral density filter in the THz range.

The C-Terminal Domain of the MutL Homolog from Neisseria gonorrhoeae Forms an Inverted Homodimer

The mismatch repair (MMR) pathway serves to maintain the integrity of the genome by removing mispaired bases from the newly synthesized strand. In E. coli, MutS, MutL and MutH coordinate to discriminate the daughter strand through a mechanism involving lack of methylation on the new strand. This facilitates the creation of a nick by MutH in the daughter strand to initiate mismatch repair. Many bacteria and eukaryotes, including humans, do not possess a homolog of MutH. Although the exact strategy for strand discrimination in these organisms is yet to be ascertained, the required nicking endonuclease activity is resident in the C-terminal domain of MutL. This activity is dependent on the integrity of a conserved metal binding motif. Unlike their eukaryotic counterparts, MutL in bacteria like Neisseria exist in the form of a homodimer. Even though this homodimer would possess two active sites, it still acts a nicking endonuclease. Here, we present the crystal structure of the C-terminal domain (CTD) of the MutL homolog of Neisseria gonorrhoeae (NgoL) determined to a resolution of 2.4 A. The structure shows that the metal binding motif exists in a helical configuration and that four of the six conserved motifs in the MutL family, including the metal binding site, localize together to form a composite active site. NgoL-CTD exists in the form of an elongated inverted homodimer stabilized by a hydrophobic interface rich in leucines. The inverted arrangement places the two composite active sites in each subunit on opposite lateral sides of the homodimer. Such an arrangement raises the possibility that one of the active sites is occluded due to interaction of NgoL with other protein factors involved in MMR. The presentation of only one active site to substrate DNA will ensure that nicking of only one strand occurs to prevent inadvertent and deleterious double stranded cleavage.

Enhanced photodynamic effect of cobalt(III) dipyridophenazine complex on thyrotropin receptor expressing HEK293 cells

Ternary cobalt(III) complexes CoL(B)] (1-3) of a trianionic tetradentate phenolate-based ligand (L) and phenanthroline bases (B), viz. 1,10-phenanthroline (phen in 1), dipyridoquinoxaline (dpq in 2) and dipyridophenazine (dppz in 3) are synthesized, characterized from X-ray crystallographic, analytical and spectral techniques, and their utility in photodynamic therapy (PDT) of thyroid diseases caused by TSH receptor dysfunction is probed. The complexes display a visible spectral band within the PDT spectral window at similar to 690 nm. Photodynamic potential was estimated through DNA cleavage activity of the dpq and dppz complexes in UV-A light of 365 nm and red light of 676 nm. The reactions proceed via the hydroxyl radical pathway. The complexes retain their DNA photocleavage activity in red light under anaerobic conditions, a situation normally prevails in hypoxic tumor core. Investigation into the photocytotoxic potential of these complexes showed that the dppz complex 3 is approximately 4-fold more active in the HEK293 cells expressing human thyrotropin receptor (HEK293-hTSHR) than in the parental cell line and has an insignificant effect on an unrelated human cervical carcinoma cell line (HeLa). Photoexcitation of complex 3 in HEK293-hTSHR cells leads to damage hTSHR as evidenced from the decrease in cAMP formation both in absence and presence of hTSH and decrease in the TSHR immunofluorescence with a concomitant cytoplasmic translocation of the membrane protein, cadherin. The involvement of hTSHR is evidenced from the ability of complex 3 to bind to the extracellular domain of hTSHR (hTSHR-ECD) with a K-d value of 81 nM and from the photocleavage of hTSHR-ECD.

Src Homology 3-interacting Domain of Rv1917c of Mycobacterium tuberculosis Induces Selective Maturation of Human Dendritic Cells by Regulating PI3K-MAPK-NF-kappa B Signaling and Drives Th2 Immune Responses

Mycobacterium tuberculosis, an etiological agent of pulmonary tuberculosis, causes significant morbidity and mortality worldwide. Pathogenic mycobacteria survive in the host by subverting host innate immunity. Dendritic cells (DCs) are professional antigen-presenting cells that are vital for eliciting immune responses to infectious agents, including pathogenic mycobacteria. DCs orchestrate distinct Th responses based on the signals they receive. In this perspective, deciphering the interactions of the proline-glutamic acid/proline-proline-glutamic acid (PE/PPE) family of proteins of M. tuberculosis with DCs assumes significant pathophysiological attributes. In this study, we demonstrate that Rv1917c (PPE34), a representative member of the proline-proline-glutamic-major polymorphic tandem repeat family, interacts with TLR2 and triggers functional maturation of human DCs. Signaling perturbations implicated a critical role for integrated cross-talk among PI3K-MAPK and NF-kappa B signaling cascades in Rv1917c-induced maturation of DCs. However, this maturation of DCs was associated with a secretion of high amounts of anti-inflammatory cytokine IL-10, whereas Th1-polarizing cytokine IL-12 was not induced. Consistent with these results, Rv1917c-matured DCs favored secretion of IL-4, IL-5, and IL-10 from CD4(+) T cells and contributed to Th2-skewed cytokine balance ex vivo in healthy individuals and in patients with pulmonary tuberculosis. Interestingly, the Rv1917c-skewed Th2 immune response involved induced expression of cyclooxygenase-2 (COX-2) in DCs. Taken together, these results indicate that Rv1917c facilitates a shift in the ensuing immunity toward the Th2 phenotype and could aid in immune evasion by mycobacteria.

Molecular studies on LIM-domain protein CRP1

Various intrinsic and external factors are constantly attacking the cells causing damage to DNA and to other cellular structures. Cells in turn have evolved with different kinds of mechanisms to protect against the attacks and to repair the damage. Ultraviolet radiation (UVR) is one of the major environmental genotoxic carcinogens that causes inflammation, mutations, immunosuppression, accelerated aging of the skin and skin cancers. Epidermis is the outermost layer of the skin consisting mostly of keratinocytes, whose primary function is to protect the skin against e.g. UV radiation. LIM domain proteins are a group of proteins involved in regulation of cell growth, damage signalling, cell fate determination and signal transduction. Despite their two zinc fingers, LIM domains do not bind to DNA, but rather mediate protein-protein interactions and function as modular protein binding interfaces. We initially identified CSRP1 as UVR-regulated transcript by using expression profiling. Here we have further studied the regulation and function of CRP1, a representative of cysteine rich protein- family consisting of two LIM domains. We find that CRP1 is increased by UVR in primary human keratinocytes and in normal human skin fibroblasts. Ectopic expression of CRP1 protected the cells against UVR and provided a survival advantage, whereas silencing of CRP1 rendered the cells more photosensitive. Actinic keratosis is a premalignant lesion of skin caused by excess exposure to sunlight and sunburn, which may lead to formation of squamous cell carcinoma. The expression of CRP1 was increased in basal keratinocytes of Actinic keratosis patient specimens suggesting that CRP1 may be increased by constant exposure to UVR and may provide survival advantage for the cells also in vivo. In squamous cell carcinoma, CRP1 was only expressed in the fibroblasts surrounding the tumour. Moreover, we found that ectopic expression of CRP1 suppresses cell proliferation. Transforming growth factor beta (TGFbeta) is a multifunctional cytokine that regulates several functions in cell including growth, apoptosis and differentiation, and plays important roles in pathological disorders like cancer and fibrosis. We found that TGFbeta-signalling pathway regulates CRP1 at protein, but not at transcriptional level. The increase was mediated both through Smad and non-Smad signalling pathways involving MAPK/p38. Furthermore, we found that TGFbeta-mediated increase in CRP1 was associated with myofibroblast differentiation, and that CRP1 was significantly more expressed in idiopathic pulmonary fibrosis as compared to normal lung specimens. Since cell contractility is a distinct feature of myofibroblasts, and CRP1 is associated with actin cytoskeleton, we studied the role of CRP1 in cell contractility. CRP1 was found to localize to stress fibres that mediate contractility and to mediate myofibroblast contraction. These studies identify CRP1 as a stress responsive and cytokine regulated cytoskeletal protein that participates in pathological processes involved in fibrotic diseases and cancer.

Acute toxicity of methyl isocyanate in rabbit: In vitro and in vivo effects on rabbit erythrocyte membrane

Methyl isocyanate (MIC) interaction with the rabbit erythrocyte membrane increased the fluidity of the membrane and decreased the osmotic fragility of erythrocytes both in vitro and in vivo in rabbits intoxicated with MIC subcutaneously. MIC inhibited both acetylcholinesterase (AChE) and adenosine triphosphatase (ATPase) activities of erythrocytes dose-dependently in vitro, while in vivo a decreased trend in ATPase activity with unaltered AChE activity was observed. MIC also caused significant decrease in plasma sodium level with corresponding increase in potassium level in rabbits. The observed effects are due to MIC, per se, as the hydrolysis products of MIC, methylamine and N,Nprime-dimethylurea did not affect the erythrocyte fluidity and enzymes activities both in vitro and in vivo while they increased the osmotic fragility of erythrocytes in vivo in rabbits administered subcutaneously in equimolar concentration to MIC dosage. Inhibition of Na+-K+-dependent ATPase with altered permeability to cations and also probably water transport of plasma membrane due to MIC interaction are envisaged.

Hantavirus infection: Insights into entry, assembly and pathogenesis

Hantaviruses (family Bunyaviridae, genus Hantavirus) are enveloped viruses incorporating a segmented, negative-sense RNA genome. Each hantavirus is carried by its specific host, either a rodent or an insectivore (shrew), in which the infection is asymptomatic and persistent. In humans, hantaviruses cause Hemorrhagic fever with renal syndrome (HFRS) in Eurasia and Hantavirus cardiopulmonary syndrome (HCPS) in the Americas. In Finland, Puumala virus (genus Hantavirus) is the causative agent of NE, a mild form of HFRS. The HFRS-type diseases are often associated with renal failure and proteinuria that might be mechanistically explained by infected kidney tubular cell degeneration in patients. Previously, it has been shown that non-pathogenic hantavirus, Tula virus (TULV), could cause programmed cell death, apoptosis, in cell cultures. This suggested that the infected kidney tubular degeneration could be caused directly by virus replication. In the first paper of this thesis the molecular mechanisms involved in TULV-induced apoptosis was further elucidated. A virus replication-dependent down-regulation of ERK1/2, concomitantly with the induced apoptosis, was identified. In addition, this phenomenon was not restricted to TULV or to non-pathogenic hantaviruses in general since also a pathogenic hantavirus, Seoul virus, could inhibit ERK1/2 activity. Hantaviruses consist of membrane-spanning glycoproteins Gn and Gc, RNA-dependent RNA polymerase (L protein) and nucleocapsid protein N, which encapsidates the viral genome, and thus forms the ribonucleoprotein (RNP). Interaction between the cytoplasmic tails of viral glycoproteins and RNP is assumed to be the only means how viral genetic material is incorporated into infectious virions. In the second paper of this thesis, it was shown by immunoprecipitation that viral glycoproteins and RNP interact in the purified virions. It was further shown that peptides derived from the cytoplasmic tails (CTs) of both Gn and Gc could bind RNP and recombinant N protein. In the fourth paper the cytoplamic tail of Gn but not Gc was shown to interact with genomic RNA. This interaction was probably rather unspecific since binding of Gn-CT with unrelated RNA and even single-stranded DNA were also observed. However, since the RNP consists of both N protein and N protein-encapsidated genomic RNA, it is possible that the viral genome plays a role in packaging of RNPs into virions. On the other hand, the nucleic acid-binding activity of Gn may have importance in the synthesis of viral RNA. Binding sites of Gn-CT with N protein or nucleic acids were also determined by peptide arrays, and they were largely found to overlap. The Gn-CT of hantaviruses contain a conserved zinc finger (ZF) domain with an unknown function. Some viruses need ZFs in entry or post-entry steps of the viral life cycle. Cysteine residues are required for the folding of ZFs by coordinating zinc-ions, and alkylation of these residues can affect virus infectivity. In the third paper, it was shown that purified hantavirions could be inactivated by treatment with cysteine-alkylating reagents, especially N-ethyl maleimide. However, the effect could not be pin-pointed to the ZF of Gn-CT since also other viral proteins reacted with maleimides, and it was, therefore, impossible to exclude the possibility that other cysteines besides those that were essential in the formation of ZF are required for hantavirus infectivity.