Magmas functions as a ROS regulator and provides cytoprotection against oxidative stress-mediated damages

Redox imbalance generates multiple cellular damages leading to oxidative stress-mediated pathological conditions such as neurodegenerative diseases and cancer progression. Therefore, maintenance of reactive oxygen species (ROS) homeostasis is most important that involves well-defined antioxidant machinery. In the present study, we have identified for the first time a component of mammalian protein translocation machinery Magmas to perform a critical ROS regulatory function. Magmas overexpression has been reported in highly metabolically active tissues and cancer cells that are prone to oxidative damage. We found that Magmas regulates cellular ROS levels by controlling its production as well as scavenging. Magmas promotes cellular tolerance toward oxidative stress by enhancing antioxidant enzyme activity, thus preventing induction of apoptosis and damage to cellular components. Magmas enhances the activity of electron transport chain (ETC) complexes, causing reduced ROS production. Our results suggest that J-like domain of Magmas is essential for maintenance of redox balance. The function of Magmas as a ROS sensor was found to be independent of its role in protein import. The unique ROS modulatory role of Magmas is highlighted by its ability to increase cell tolerance to oxidative stress even in yeast model organism. The cytoprotective capability of Magmas against oxidative damage makes it an important candidate for future investigation in therapeutics of oxidative stress-related diseases.

Mycobacterium tuberculosis DinG Is a Structure-specific Helicase That Unwinds G4 DNA IMPLICATIONS FOR TARGETING G4 DNA AS A NOVEL THERAPEUTIC APPROACH

The significance of G-quadruplexes and the helicases that resolve G4 structures in prokaryotes is poorly understood. The Mycobacterium tuberculosis genome is GC-rich and contains >10,000 sequences that have the potential to form G4 structures. In Escherichia coli, RecQ helicase unwinds G4 structures. However, RecQ is absent in M. tuberculosis, and the helicase that participates in G4 resolution in M. tuberculosis is obscure. Here, we show that M. tuberculosis DinG (MtDinG) exhibits high affinity for ssDNA and ssDNA translocation with a 5' -> 3' polarity. Interestingly, MtDinG unwinds overhangs, flap structures, and forked duplexes but fails to unwind linear duplex DNA. Our data with DNase I footprinting provide mechanistic insights and suggest that MtDinG is a 5' -> 3' polarity helicase. Notably, in contrast to E. coli DinG, MtDinG catalyzes unwinding of replication fork and Holliday junction structures. Strikingly, we find that MtDinG resolves intermolecular G4 structures. These data suggest that MtDinG is a multifunctional structure-specific helicase that unwinds model structures of DNA replication, repair, and recombination as well as G4 structures. We finally demonstrate that promoter sequences of M. tuberculosis PE_PGRS2, mce1R, and moeB1 genes contain G4 structures, implying that G4 structures may regulate gene expression in M. tuberculosis. We discuss these data and implicate targeting G4 structures and DinG helicase in M. tuberculosis could be a novel therapeutic strategy for culminating the infection with this pathogen.

Salmonella methylglyoxal detoxification by STM3117-encoded lactoylglutathione lyase affects virulence in coordination with Salmonella pathogenicity island 2 and phagosomal acidification

Intracellular pathogens such as Salmonella enterica serovar Typhimurium (S. Typhimurium) manipulate their host cells through the interplay of various virulence factors. A multitude of such virulence factors are encoded on the genome of S. Typhimurium and are usually organized in pathogenicity islands. The virulence-associated genomic stretch of STM3117-3120 has structural features of pathogenicity islands and is present exclusively in non-typhoidal serovars of Salmonella. It encodes metabolic enzymes predicted to be involved in methylglyoxal metabolism. STM3117-encoded lactoylglutathione lyase significantly impacts the proliferation of intracellular Salmonella. The deletion mutant of STM3117 (Delta lgl) fails to grow in epithelial cells but hyper-replicates in macrophages. This difference in proliferation outcome was the consequence of failure to detoxify methylglyoxal by Delta lgl, which was also reflected in the form of oxidative DNA damage and upregulation of kefB in the mutant. Within macrophages, the toxicity of methylglyoxal adducts elicits the potassium efflux channel (KefB) in the mutant which subsequently modulates the acidification of mutant-containing vacuoles (MCVs). The perturbation in the pH of the MCV milieu and bacterial cytosol enhances the Salmonella pathogenicity island 2 translocation in Delta lgl, increasing its net growth within macrophages. In epithelial cells, however, the maturation of Delta lgl-containing vacuoles were affected as these non-phagocytic cells maintain less acidic vacuoles compared to those in macrophages. Remarkably, ectopic expression of Toll-like receptors 2 and 4 on epithelial cells partially restored the survival of Delta lgl. This study identified a novel metabolic enzyme in S. Typhimurium whose activity during intracellular infection within a given host cell type differentially affected the virulence of the bacteria.

Imaging cellular signalling: many `moving tales' in MAP kinase odyssey

Cellular signalling events are at the core of every adaptive response. Signalling events link environmental changes to physiological responses, consequently allowing cellular and organismal sustenance and survival. Classical approaches to study cellular signalling have relied on a variety of cell disruptive techniques which yield limited kinetic information, while the underlying events are much more complex. In this article, we discuss how modern live cell imaging microscopy has found increasing utilization in revealing spatio temporal dynamics of various signalling pathways. Utilizing the well studied mitogen-activated protein kinase (MAPK) signalling cascade as a template, the design, construction and utilization of `mobile' (translocation proficient) biosensors, suitable for studying MAPK signalling in living cells are described in detail. Experimental setup and results obtained from these biosensors, based on different proteins involved in the MAPK signalling cascade, have been described along with the setup of a microscope optimal for live cell imaging applications. Utilizing the ability to activate or deactivate signalling pathways using defined activators and specific pharmacological inhibitors, we also show how these sensors can yield unique spatial and temporal kinetic information of signalling in living cells.

Characterization of Precursor PfHsp60 in Plasmodium falciparum Cytosol during Its Asexual Development in Human Erythrocytes

Mitochondrial heat shock protein 60 (Hsp60) is a nuclear encoded gene product that gets post-translationally translocated into the mitochondria. Using multiple approaches such as immunofluorescence experiments, isoelectric point analysis with two-dimensional gel electrophoresis, and mass spectrometric identification of the signal peptide, we show that Hsp60 from Plasmodium falciparum (PfHsp60) accumulates in the parasite cytoplasm during the ring, trophozoite, and schizont stages of parasite development before being imported into the parasite mitochondria. Using co-immunoprecipitation experiments with antibodies specific to cytoplasmic PfHsp90, PfHsp70-1, and PfHsp60, we show association of precursor PfHsp60 with cytoplasmic chaperone machinery. Metabolic labeling involving pulse and chase indicates translocation of the precursor pool into the parasite mitochondrion during chase. Analysis of results obtained with Geldanamycin treatment confirmed precursor PfHsp60 to be one of the clients for PfHsp90. Cytosolic chaperones bind precursor PfHsp60 prior to its import into the mitochondrion of the parasite. Our data suggests an inefficient co-ordination in the synthesis and translocation of mitochondrial PfHsp60 during asexual growth of malaria parasite in human erythrocytes.

New Oncogenic Drivers in Hepatocellular Carcinoma: Role of the RNA Binding Protein Hu antigen R

156 p. : graf.

Novel mechanisms for regulation of cell survival and migration by ceramide 1-phosphate

201 p. : gráf.

NMDA modulates oligodendrocyte differentiation of subventricular zone cells through PKC activation

7 p.

Calpain-Mediated Processing of Adenylate Cyclase Toxin Generates a Cytosolic Soluble Catalytically Active N-Terminal Domain

Bordetella pertussis, the whooping cough pathogen, secretes several virulence factors among which adenylate cyclase toxin (ACT) is essential for establishment of the disease in the respiratory tract. ACT weakens host defenses by suppressing important bactericidal activities of the phagocytic cells. Up to now, it was believed that cell intoxication by ACT was a consequence of the accumulation of abnormally high levels of cAMP, generated exclusively beneath the host plasma membrane by the toxin N-terminal catalytic adenylate cyclase (AC) domain, upon its direct translocation across the lipid bilayer. Here we show that host calpain, a calcium-dependent Cys-protease, is activated into the phagocytes by a toxin-triggered calcium rise, resulting in the proteolytic cleavage of the toxin N-terminal domain that releases a catalytically active "soluble AC''. The calpain-mediated ACT processing allows trafficking of the "soluble AC'' domain into subcellular organella. At least two strategic advantages arise from this singular toxin cleavage, enhancing the specificity of action, and simultaneously preventing an indiscriminate activation of cAMP effectors throughout the cell. The present study provides novel insights into the toxin mechanism of action, as the calpain-mediated toxin processing would confer ACT the capacity for a space- and time-coordinated production of different cAMP "pools'', which would play different roles in the cell pathophysiology.

Resveratrol prevents inflammation-dependent hepatic melanoma metastasis by inhibiting the secretion and effects of interleukin-18

Background: Implantation and growth of metastatic cancer cells at distant organs is promoted by inflammation-dependent mechanisms. A hepatic melanoma metastasis model where a majority of metastases are generated via interleukin-18-dependent mechanisms was used to test whether anti-inflammatory properties of resveratrol can interfere with mechanisms of metastasis. Methods: Two experimental treatment schedules were used: 1) Mice received one daily oral dose of 1 mg/kg resveratrol after cancer cell injection and the metastasis number and volume were determined on day 12. 2) Mice received one daily oral dose of 1 mg/kg resveratrol along the 5 days prior to the injection of cancer cells and both interleukin-18 (IL-18) concentration in the hepatic blood and microvascular retention of luciferase-transfected B16M cells were determined on the 18(th) hour. In vitro, primary cultured hepatic sinusoidal endothelial cells were treated with B16M-conditioned medium to mimic their in vivo activation by tumor-derived factors and the effect of resveratrol on IL-18 secretion, on vascular cell adhesion molecule-1 (VCAM-1) expression and on tumor cell adhesion were studied. The effect of resveratrol on melanoma cell activation by IL-18 was also studied. Results: Resveratrol remarkably inhibited hepatic retention and metastatic growth of melanoma cells by 50% and 75%, respectively. The mechanism involved IL-18 blockade at three levels: First, resveratrol prevented IL-18 augmentation in the blood of melanoma cell-infiltrated livers. Second, resveratrol inhibited IL-18-dependent expression of VCAM-1 by tumor-activated hepatic sinusoidal endothelium, preventing melanoma cell adhesion to the microvasculature. Third, resveratrol inhibited adhesion-and proliferation-stimulating effects of IL-18 on metastatic melanoma cells through hydrogen peroxide-dependent nuclear factor-kappaB translocation blockade on these cells. Conclusions: These results demonstrate multiple sites for therapeutic intervention using resveratrol within the prometastatic microenvironment generated by tumor-induced hepatic IL-18, and suggest a remarkable effect of resveratrol in the prevention of inflammation-dependent melanoma metastasis in the liver.

Negative regulation of transcription factors by Srb10 cyclin-dependent kinase

The ubiquitin-dependent proteolytic pathway plays an important role in a broad array of cellular processes, inducting cell cycle control and transcription. Biochemical analysis of the ubiquitination of Sic1, the B-type cyclin-dependent kinase (CDK) inhibitor in budding yeast helped to define a ubiquitin ligase complex named SCF^cdc4 (for Skp1, Cdc53/cullin, F-box protein). We found that besides Sic1, the CDK inhibitor Far1 and the replication initiation protein Cdc6 are also substrates of SCF^cdc4 in vitro. A common feature in the ubiquitination of the cell cycle SCF^cdc4 substrates is that they must be phosphorylated by the major cell cycle CDK, Cdc28. Gcn4, a transcription activator involved in the general control of amino acid biosynthesis, is rapidly degraded in an SCF^cdc4-dependent manner in vivo. We have focused on this substrate to investigate the generality of the SCF^cdc4 pathway. Through biochemical fractionations, we found that the Srb10 CDK phosphorylates Gcn4 and thereby marks it for recognition by SCF^cdc4 ubiquitin ligase. Srb10 is a physiological regulator of Gcn4 stability because both phosphorylation and turnover of Gcn4 are diminished in srb10 mutants. Furthermore, we found that at least two different CDKs, Pho85 and Srb10, conspire to promote the rapid degradation of Gcn4 in vivo. The multistress response transcriptional regulator Msn2 is also a substrate for Srb10 and is hyperphosphorylated in an Srb10-dependent manner upon heat stress-induced translocation into the nucleus. Whereas Msn2 is cytoplasmic in resting wild type cells, its nuclear exclusion is partially compromised in srb10 mutant cells. Srb10 has been shown to repress a subset of genes in vivo, and has been proposed to inhibit transcription via phosphorylation of the C-terminal domain of RNA polymerase II. Our results suggest a general theme that Srb10 represses the transcription of specific genes by directly antagonizing the transcriptional activators.

Phospholipids and a protein-conducting channel regulate cotranslational protein targeting

Signal recognition particle (SRP) and signal recognition particle receptor (SR) are evolutionarily conserved GTPases that deliver secretory and membrane proteins to the protein-conducting channel Sec61 complex in the lipid bilayer of the endoplasmic reticulum in eukaryotes or the SecYEG complex in the inner membrane of bacteria. Unlike the canonical Ras-type GTPases, SRP and SR are activated via nucleotide-dependent heterodimerization. Upon formation of the SR•SRP targeting complex, SRP and SR undergo a series of discrete conformational changes that culminate in their reciprocal activation and hydrolysis of GTP. How the SR•SRP GTPase cycle is regulated and coupled to the delivery of the cargo protein to the protein-conducting channel at the target membrane is not well-understood. Here we examine the role of the lipid bilayer and SecYEG in regulation of the SRP-mediated protein targeting pathway and show that they serve as important biological cues that spatially control the targeting reaction.

In the first chapter, we show that anionic phospholipids of the inner membrane activate the bacterial SR, FtsY, and favor the late conformational states of the targeting complex conducive to efficient unloading of the cargo. The results of our studies suggest that the lipid bilayer acts as a spatial cue that weakens the interaction of the cargo protein with SRP and primes the complex for unloading its cargo onto SecYEG.

In the second chapter, we focus on the effect of SecYEG on the conformational states and activity of the targeting complex. While phospholipids prime the complex for unloading its cargo, they are insufficient to trigger hydrolysis of GTP and the release of the cargo from the complex. SecYEG modulates the conformation of the targeting complex and triggers the GTP hydrolysis from the complex, thus driving the targeting reaction to completion. The results of this study suggest that SecYEG is not a passive recipient of the cargo protein; rather, it actively releases the cargo from the targeting complex. Together, anionic phospholipids and SecYEG serve distinct yet complementary roles. They spatially control the targeting reaction in a sequential manner, ensuring efficient delivery and unloading of the cargo protein.

In the third chapter, we reconstitute the transfer reaction in vitro and visualize it in real time. We show that the ribosome-nascent chain complex is transferred to SecYEG via a stepwise mechanism with gradual dissolution and formation of the contacts with SRP and SecYEG, respectively, explaining how the cargo is kept tethered to the membrane during the transfer and how its loss to the cytosol is avoided.

In the fourth chapter, we examine interaction of SecYEG with secretory and membrane proteins and attempt to address the role of a novel insertase YidC in this interaction. We show that detergent-solubilized SecYEG is capable of discriminating between the nascent chains of various lengths and engages a signal sequence in a well-defined conformation in the absence of accessory factors. Further, YidC alters the conformation of the signal peptide bound to SecYEG. The results described in this chapter show that YidC affects the SecYEG-nascent chain interaction at early stages of translocation/insertion and suggest a YidC-facilitated mechanism for lateral exit of transmembrane domains from SecYEG into the lipid bilayer.

The isolation and partial biochemical analysis of Sindbis virus proteins

Recently, the amino acid sequences have been reported for several proteins, including the envelope glycoproteins of Sindbis virus, which all probably span the plasma membrane with a common topology: a large N-terminal, extracellular portion, a short region buried in the bilayer, and a short C-terminal intracellular segment. The regions of these proteins buried in the bilayer correspond to portions of the protein sequences which contain a stretch of hydrophobic amino acids and which have other common characteristics, as discussed. Reasons are also described for uncertainty, in some proteins more than others, as to the precise location of some parts of the sequence relative to the membrane.

The signal hypothesis for the transmembrane translocation of proteins is briefly described and its general applicability is reviewed. There are many proteins whose translocation is accurately described by this hypothesis, but some proteins are translocated in a different manner.

The transmembraneous glycoproteins E1 and E2 of Sindbis virus, as well as the only other virion protein, the capsid protein, were purified in amounts sufficient for biochemical analysis using sensitive techniques. The amino acid composition of each protein was determined, and extensive N-terminal sequences were obtained for E1 and E2. By these techniques E1 and E2 are indistinguishable from most water soluble proteins, as they do not contain an obvious excess of hydrophobic amino acids in their N-terminal regions or in the intact molecule.

The capsid protein was found to be blocked, and so its N-terminus could not be sequenced by the usual methods. However, with the use of a special labeling technique, it was possible to incorporate tritiated acetate into the N-terminus of the protein with good specificity, which was useful in the purification of peptides from which the first amino acids in the N-terminal sequence could be identified.

Nanomole amounts of PE2, the intracellular precursor of E2, were purified by an immuno-affinity technique, and its N-terminus was analyzed. Together with other work, these results showed that PE2 is not synthesized with an N-terminal extension, and the signal sequence for translocation is probably the N-terminal amino acid sequence of the protein. This N-terminus was found to be 80-90% blocked, also by Nacetylation, and this acetylation did not affect its function as a signal sequence. The putative signal sequence was also found to contain a glycosylated asparagine residue, but the inhibition of this glycosylation did not lead to the cleavage of the sequence.

Caracterização de alterações epigenéticas no gene JARID1C e desequilíbrios genéticos como causas do retardo mental ligado ao x de etiologia idiopática

O retardo mental (RM) é caracterizado por um funcionamento intelectual significantemente abaixo da média (QI<70). A prevalência de RM varia entre estudos epidemiológicos, sendo estimada em 2-3% da população mundial, constituindo assim, um dos mais importantes problemas de saúde pública. Há um consenso geral de que o RM é mais comum no sexo masculino, um achado atribuído às numerosas mutações nos genes encontrados no cromossomo X, levando ao retardo mental ligado ao X (RMLX). Dentre os genes presentes no cromossomo X, o Jumonji AT-rich interactive domain IC (JARID1C) foi recentemente identificado como um potencial candidato etiológico do RM, quando mutado. O JARID1C codifica uma proteína que atua como uma desmetilase da lisina 4 da histona H3 (H3K4), imprescindível para a regulação epigenética. Tão recente como a identificação do gene JARID1C, é a descoberta de que mudanças no número de cópias de sequências de DNA, caracterizadas por microdeleções e microduplicações, poderiam ser consideradas como razões funcionalmente importantes de RMLX. Atualmente, cerca de 5-10% dos casos de RM em homens são reconhecidos por ocorrerem devido a estas variações do número de cópias no cromossomo X. Neste estudo, investigamos mutações no gene JARID1C, através do rastreamento dos éxons 9, 11, 12, 13, 15 e 16, em 121 homens de famílias com RM provavelmente ligado ao X. Paralelamente, realizamos a análise da variação do número de cópias em 16 genes localizados no cromossomo X através da técnica de MLPA no mesmo grupo de pacientes. Esta metodologia consiste em uma amplificação múltipla que detecta variações no número de cópias de até 50 sequências diferentes de DNA genômico, sendo capaz de distinguir sequências que diferem em apenas um nucleotídeo. O DNA genômico foi extraído a partir de sangue periférico e as amostras foram amplificadas pela técnica de PCR, seguida da análise por sequenciamento direto. Foram identificadas três variantes na sequência do gene JARID1C entre os pacientes analisados: a variante intrônica 2243+11 G>T, que esteve presente em 67% dos pacientes, a variante silenciosa c.1794C>G e a mutação inédita nonsense c.2172C>A, ambas presentes em 0,82% dos indivíduos investigados. A análise através do MLPA revelou uma duplicação em um dos pacientes envolvendo as sondas referentes ao gene GDI1 e ao gene HUWE1. Este trabalho expande o estudo de mutações no gene JARID1C para a população brasileira ereforça a importância da triagem de mutações neste gene em homens portadores de RM familiar de origem idiopática, assim como, é primeiro relato científico relativo à investigação de variações no número de cópias de genes localizados no cromossomo X em homens brasileiros com RM, através da técnica de MLPA.

Efeito de ExoU na ativação de NF-κB e na secreção de IL-8 por células humanas infectadas por Pseudomonas aeruginosa

ExoU, uma citotoxina produzida pelo patógeno oportunista Pseudomonas aeruginosa e translocada para o citossol de células hospedeiras via sistema de secreção do tipo III, é associada à gravidade de infecções agudas. Estudos anteriores realizados em nosso laboratório relataram a potente atividade pró-inflamatória de ExoU, responsável por um intenso recrutamento de neutrófilos para o sítio de infecção. No presente trabalho, o efeito de ExoU na modulação da ativação do fator transcricional NF-κB e na regulação da expressão e da secreção da quimiocina para neutrófilos IL-8 foi avaliado em culturas de células epiteliais respiratórias e endoteliais humanas infectadas com a cepa PA103 de P. aeruginosa (produtora de ExoU) ou com a mutante deletada no gene exoU, PA103κexoU. Análises por RT-PCR semi-quantitativo mostraram que a infecção pela cepa produtora de ExoU levou ao aumento dos níveis de mRNA de IL-8, enquanto ensaios de alteração da mobilidade eletroforética (EMSA), supershift e com gene repórter mostraram que ExoU induziu a translocação nuclear do heterodímero transativador p65/p50 de NF-κB e a ativação da transcrição de genes dependente deste fator transcricional. Adicionalmente, o tratamento das culturas celulares com um inibidor de NF-κB antes da infecção bacteriana reduziu significativamente os níveis de mRNA de IL-8 e da secreção desta quimiocina. Em conjunto, estes resultados mostram que ExoU ativa NF-κB e, consequentemente, estimula a expressão e a secreção de IL-8 por células epiteliais respiratórias e células endoteliais infectadas com P. aeruginosa