Interaction entre la RNase HI et la RNase E dans le métabolisme des R-loops et la dégradation des ARNms chez Escherichia coli.

Chez la bactérie Escherichia coli, la topoisomérase I et la gyrase représentent deux topoisomérases majeures qui participent à la régulation du surenroulement de l’ADN. Celles-ci sont codées respectivement par les gènes topA et par gyrA et gyrB. Chez les mutants topA, l’excès de surenroulement négatif qui est généré en amont de la polymérase ARN lors de la phase d’élongation de la transcription de l’ADN, entraine la formation de R-loops. Les R-loops sont des hybrides ARN-ADN qui in vivo sont formés lorsque l’ARN nouvellement transcrit forme un hybride avec le brin d’ADN matrice, le brin d’ADN complémentaire demeurant sous forme simple brin. La RNase HI est une endoribonucléase codée par le gène rnhA. Elle dégrade l’ARN de R-loops, entre autres, pour empêcher l’initiation de la réplication à des sites autres que l’origine normale, oriC. Chez les mutants rnhA, on observe une réplication indépendante de l’origine oriC. Ce type de réplication appelé cSDR, pourrait donc expliquer, du moins en partie, l’inhibition de la croissance de doubles mutants topA rnhA. A l’aide de la mutagenèse au transposon Tn5, il a été possible d’isoler des suppresseurs extragéniques qui permettaient la croissance des doubles mutants topA rnhA. Plusieurs de ces suppresseurs ont le transposon inséré dans le gène codant pour la RNase E, l’endoribonucléase principale impliquée dans la dégradation des ARNms chez E. coli. La majorité des insertions se retrouvent dans la partie C-terminale de la protéine qui est impliquée dans l’assemblage d’un complexe multiprotéique appelé l’ARN dégradosome. Les résultats obtenus démontrent que ces suppresseurs diminuent le cSDR ainsi que la réponse SOS induite constitutivement en l’absence de la RNase HI. Sachant que la RNase HI est une endoribonucléase tout comme la RNase E, une collaboration entre les deux enzymes suggère que la RNase E pourrait également jouer un rôle potentiel dans le contrôle de la formation des R-loops et bien évidemment de leur retrait au sein de la cellule. À l’opposé, il est possible que la RNase HI puisse avoir comme autre fonction la prise en charge de la maturation et de la dégradation des molécules d’ARNs.

THE ROLE OF INNATE IMMUNITY IN SEPTIC ACUTE KIDNEY INJURIES

Acute kidney injury (AKI) is an important clinical syndrome characterized by abnormalities in the hydroelectrolytic balance. Because of high rates of morbidity and mortality (from 15% to 60%) associated with AKI, the study of its pathophysiology is critical in searching for clinical targets and therapeutic strategies. Severe sepsis is the major cause of AKI. The host response to sepsis involves an inflammatory response, whereby the pathogen is initially sensed by innate immune receptors (pattern recognition receptors [PRRs]). When it persists, this immune response leads to secretion of proinflammatory products that induce organ dysfunction such as renal failure and consequently increased mortality. Moreover, the injured tissue releases molecules resulting from extracellular matrix degradation or dying cells that function as alarmines, which are recognized by PRR in the absence of pathogens in a second wave of injury. Toll-like receptors (TLRs) and NOD-like receptors (NLRs) are the best characterized PRRs. They are expressed in many cell types and throughout the nephron. Their activation leads to translocation of nuclear factors and synthesis of proinflammatory cytokines and chemokines. TLRs` signaling primes the cells for a robust inflammatory response dependent on NLRs; the interaction of TLRs and NLRs gives rise to the multiprotein complex known as the inflammasome, which in turn activates secretion of mature interleukin 1 beta and interleukin 18. Experimental data show that innate immune receptors, the inflammasome components, and proinflammatory cytokines play crucial roles not only in sepsis, but also in organ-induced dysfunction, especially in the kidneys. In this review, we discuss the significance of the innate immune receptors in the development of acute renal injury secondary to sepsis.

Estudo das interações entre a proteína TRF de Leishmania amazonensis (LaTRF) e suas possíveis parcerias

Pós-graduação em Ciências Biológicas (Genética) - IBB

Gamma-secretase modulators and inhibitors in Alzheimer's disease: influence of genetic background on efficacy and mechanism of action

According to the amyloid hypothesis, Alzheimer’s disease (AD) is caused by aberrant production or clearance of the amyloid-β (Aβ) peptides, and in particular of the longer more aggregation-prone Aβ42. The Aβ peptides are generated through successive proteolytic cleavage of the amyloid precursor protein (APP) by the β-site APP cleaving enzyme (BACE) and γ-secretase. γ-secretase produces Aβ peptides with variable C-termini ranging from Aβ34 to Aβ48, presumably by sequential trimming of longer into shorter peptides. γ-secretase is a multiprotein complex consisting of at least four different proteins and the presenilin proteins (PS1 or PS2) contain the catalytic center of the complex. In 2001 several non-steroidal anti-inflammatory drugs were identified as the founding members of a new class of γ-secretase modulators (GSMs) that can selectively reduce production of Aβ42. Concomitantly, these GSMs increase Aβ38 production indicating closely coordinated generation of Aβ42 and Aβ38 and a potential precursor-product relationship between these peptides. GSMs seem to exert their activity by direct modulation of γ-secretase. Support for this hypothesis is drawn from the finding that some PS mutations associated with early-onset familial AD (FAD) can modulate the cellular response to GSMs and to γ-secretase inhibitors (GSIs), which inhibit production of all Aβ peptides and are known to directly interact with PS. A particularly interesting FAD PS mutation is PS1-ΔExon9, a complex deletion mutant that blocks endoproteolysis of PS1 and renders cells completely non-responsive to GSMs. Studies presented in this thesis show that the diminished response of PS1-ΔExon9 to GSMs is mainly caused by its lack of endoproteolytic cleavage. Furthermore, we were able to demonstrate that a reduced response to GSMs and GSIs is not limited to PS1-ΔExon9 but is a common effect of aggressive FAD-associated PS1 mutations. Surprisingly, we also found that while the Aβ42 response to GSMs is almost completely abolished by these PS1 mutations, the accompanying Aβ38 increase was indistinguishable to wild-type PS1. Finally, the reduced response to GSIs was confirmed in a mouse model with transgenic expression of an aggressive FAD-associated PS1 mutation as a highly potent GSI failed to reduce Aβ42 levels in brain of these mice. Taken together, our findings provide clear evidence for independent generation of Aβ42 and Aβ38 peptides, and argue that the sequential cleavage model might be an oversimplification of the molecular mechanism of γ-secretase. Most importantly, our results highlight the significance of genetic background in drug discovery efforts aimed at γ-secretase, and indicate that the use of cellular models with transgenic expression of FAD-associated PS mutations might confound studies of the potency and efficacy of GSMs and GSIs. Therefore, such models should be strictly avoided in the ongoing preclinical development of these promising and potentially disease-modifying therapeutics for AD.

Proteasome inhibitor (MG132) rescues Nav1.5 protein content and the cardiac sodium current in dystrophin-deficient mdx (5cv) mice

The cardiac voltage-gated sodium channel, Nav1.5, plays a central role in cardiac excitability and impulse propagation and associates with the dystrophin multiprotein complex at the lateral membrane of cardiomyocytes. It was previously shown that Nav1.5 protein content and the sodium current (l Na) were both decreased in cardiomyocytes of dystrophin-deficient mdx (5cv) mice. In this study, wild-type and mdx (5cv) mice were treated for 7 days with the proteasome inhibitor MG132 (10 μg/Kg/24 h) using implanted osmotic mini pumps. MG132 rescued both the total amount of Nav1.5 protein and l Na but, unlike in previous studies, de novo expression of dystrophin was not observed in skeletal or cardiac muscle. This study suggests that the reduced expression of Nav1.5 in dystrophin-deficient cells is dependent on proteasomal degradation.

Development of an in vitro mRNA decay system for Escherichia coli: Poly(A) polymerase I is necessary to trigger degradation

Using a novel Escherichia coli in vitro decay system in which polysomes are the source of both enzymes and mRNA, we demonstrate a requirement for poly(A) polymerase I (PAP I) in mRNA turnover. The in vitro decay of two different mRNAs (trxA and lpp) is triggered by the addition of ATP only when polysomes are prepared from a strain carrying the wild-type gene for PAP I (pcnB+). The relative decay rates of these two messages are similar in vitro and in vivo. Poly(A) tails are formed on both mRNAs, but no poly(A) tails are detected on the 3′ end of mature 23S rRNA. The size distribution of poly(A) tails generated in vitro, averaging 50 nt in length, is comparable to that previously reported in vivo. PAP I activity is associated exclusively with the polysomes. Exogenously added PAP I does not restore mRNA decay to PAP I− polysomes, suggesting that, in vivo, PAP I may be part of a multiprotein complex. The potential of this in vitro system for analyzing mRNA decay in E. coli is discussed.

Pontin52, an interaction partner of β-catenin, binds to the TATA box binding protein

β-catenin, the vertebrate homolog of the Drosophila Armadillo protein, has been shown to have dual cellular functions, as a component of both the cadherin-catenin cell adhesion complex and the Wnt signaling pathway. At Wnt signaling, β-catenin becomes stabilized in the cytoplasm and subsequently available for interaction with transcription factors of the lymphocyte enhancer factor-1/T-cell factor family, resulting in a nuclear localization of β-catenin. Although β-catenin does not bind DNA directly, its carboxyl- and amino-terminal regions exhibit a transactivating activity still not well understood molecularly. Here we report the identification of an interaction partner of β-catenin, a nuclear protein designated Pontin52. Pontin52 binds β-catenin in the region of Armadillo repeats 2–5 and, more importantly, also binds the TATA box binding protein. We provide evidence for an in vivo multiprotein complex composed of Pontin52, β-catenin, and lymphocyte enhancer factor-1/T-cell factor. Our results suggest involvement of Pontin52 in the nuclear function of β-catenin.

High-Copy Suppressor Analysis Reveals a Physical Interaction between Sec34p and Sec35p, a Protein Implicated in Vesicle Docking

A temperature-sensitive mutant, sec34-2, is defective in the late stages of endoplasmic reticulum (ER)-to-Golgi transport. A high-copy suppressor screen that uses the sec34-2 mutant has resulted in the identification of the SEC34 structural gene and a novel gene called GRP1. GRP1 encodes a previously unidentified hydrophilic yeast protein related to the mammalian Golgi protein golgin-160. Although GRP1 is not essential for growth, the grp1Δ mutation displays synthetic lethal interactions with several mutations that result in ER accumulation and a block in the late stages of ER-to-Golgi transport, but not with those that block the budding of vesicles from the ER. Our findings suggest that Grp1p may facilitate membrane traffic indirectly, possibly by maintaining Golgi function. In an effort to identify genes whose products physically interact with Sec34p, we also tested the ability of overexpressed SEC34 to suppress known secretory mutations that block vesicular traffic between the ER and the Golgi. This screen revealed that SEC34 specifically suppresses sec35-1. SEC34 encodes a hydrophilic protein of ∼100 kDa. Like Sec35p, which has been implicated in the tethering of ER-derived vesicles to the Golgi, Sec34p is predominantly soluble. Sec34p and Sec35p stably associate with each other to form a multiprotein complex of ∼480 kDa. These data indicate that Sec34p acts in conjunction with Sec35p to mediate a common step in vesicular traffic.

IFN consensus sequence binding protein potentiates STAT1-dependent activation of IFNγ-responsive promoters in macrophages

IFNγ, once called the macrophage-activating factor, stimulates many genes in macrophages, ultimately leading to the elicitation of innate immunity. IFNγ's functions depend on the activation of STAT1, which stimulates transcription of IFNγ-inducible genes through the GAS element. The IFN consensus sequence binding protein (icsbγ or IFN regulatory factor 8), encoding a transcription factor of the IFN regulatory factor family, is one of such IFNγ-inducible genes in macrophages. We found that macrophages from ICSBP−/− mice were defective in inducing some IFNγ-responsive genes, even though they were capable of activating STAT1 in response to IFNγ. Accordingly, IFNγ activation of luciferase reporters fused to the GAS element was severely impaired in ICSBP−/− macrophages, but transfection of ICSBP resulted in marked stimulation of these reporters. Consistent with its role in activating IFNγ-responsive promoters, ICSBP stimulated reporter activity in a GAS-specific manner, even in the absence of IFNγ treatment, and in STAT1 negative cells. Indicative of a mechanism for this stimulation, DNA affinity binding assays revealed that endogenous ICSBP was recruited to a multiprotein complex that bound to GAS. These results suggest that ICSBP, when induced by IFNγ through STAT1, in turn generates a second wave of transcription from GAS-containing promoters, thereby contributing to the elicitation of IFNγ's unique activities in immune cells.

The serine and threonine residues in the Ig-α cytoplasmic tail negatively regulate immunoreceptor tyrosine-based activation motif-mediated signal transduction

The B cell antigen receptor (BCR) is a multiprotein complex consisting of the membrane-bound Ig molecule and the Ig-α/Ig-β heterodimer. On BCR engagement, Ig-α and Ig-β become phosphorylated not only on tyrosine residues of the immunoreceptor tyrosine-based activation motif but also on serine and threonine residues. We have mutated all serine and threonine residues in the Ig-α tail to alanine and valine, respectively. The mutated Ig-α sequence was expressed either as a single-chain Fv/Ig-α molecule or in the context of the complete BCR. In both cases, the mutated Ig-α showed a stronger tyrosine phosphorylation than the wild-type Ig-α and initiated increased signaling on stimulation. These findings suggest that serine/threonine kinases can negatively regulate signal transduction from the BCR.

Histones H3 and H4 are components of upstream activation factor required for the high-level transcription of yeast rDNA by RNA polymerase I

RNA polymerase I (Pol I) transcription in the yeast Saccharomyces cerevisiae is greatly stimulated in vivo and in vitro by the multiprotein complex, upstream activation factor (UAF). UAF binds tightly to the upstream element of the rDNA promoter, such that once bound (in vitro), UAF does not readily exchange onto a competing template. Of the polypeptides previously identified in purified UAF, three are encoded by genes required for Pol I transcription in vivo: RRN5, RRN9, and RRN10. Two others, p30 and p18, have remained uncharacterized. We report here that the N-terminal amino acid sequence, its mobility in gel electrophoresis, and the immunoreactivity of p18 shows that it is histone H3. In addition, histone H4 was found in UAF, and myc-tagged histone H4 could be used to affinity-purify UAF. Histones H2A and H2B were not detectable in UAF. These results suggest that histones H3 and H4 probably account for the strong binding of UAF to DNA and may offer a means by which general nuclear regulatory signals could be transmitted to Pol I.

The LIM-domain binding protein Ldb1 and its partner LMO2 act as negative regulators of erythroid differentiation

The nuclear LIM domain protein LMO2, a T cell oncoprotein, is essential for embryonic erythropoiesis. LIM-only proteins are presumed to act primarily through protein-protein interactions. We, and others, have identified a widely expressed protein, Ldb1, whose C-terminal 76-residues are sufficient to mediate interaction with LMO2. In murine erythroleukemia cells, the endogenous Lbd1 and LMO2 proteins exist in a stable complex, whose binding affinity appears greater than that between LMO2 and the bHLH transcription factor SCL. However, Ldb1, LMO2, and SCL/E12 can assemble as a multiprotein complex on a consensus SCL binding site. Like LMO2, the Ldb1 gene is expressed in fetal liver and erythroid cell lines. Forced expression of Ldb1 in G1ER proerythroblast cells inhibited cellular maturation, a finding compatible with the decrease in Ldb1 gene expression that normally occurs during erythroid differentiation. Overexpression of the LMO2 gene also inhibited erythroid differentiation. Our studies demonstrate a function for Ldb1 in hemopoietic cells and suggest that one role of the Ldb1/LMO2 complex is to maintain erythroid precursors in an immature state.

A comprehensive two-hybrid analysis to explore the yeast protein interactome

Protein–protein interactions play crucial roles in the execution of various biological functions. Accordingly, their comprehensive description would contribute considerably to the functional interpretation of fully sequenced genomes, which are flooded with novel genes of unpredictable functions. We previously developed a system to examine two-hybrid interactions in all possible combinations between the ≈6,000 proteins of the budding yeast Saccharomyces cerevisiae. Here we have completed the comprehensive analysis using this system to identify 4,549 two-hybrid interactions among 3,278 proteins. Unexpectedly, these data do not largely overlap with those obtained by the other project [Uetz, P., et al. (2000) Nature (London) 403, 623–627] and hence have substantially expanded our knowledge on the protein interaction space or interactome of the yeast. Cumulative connection of these binary interactions generates a single huge network linking the vast majority of the proteins. Bioinformatics-aided selection of biologically relevant interactions highlights various intriguing subnetworks. They include, for instance, the one that had successfully foreseen the involvement of a novel protein in spindle pole body function as well as the one that may uncover a hitherto unidentified multiprotein complex potentially participating in the process of vesicular transport. Our data would thus significantly expand and improve the protein interaction map for the exploration of genome functions that eventually leads to thorough understanding of the cell as a molecular system.

Considerations of transcriptional control mechanisms: Do TFIID–core promoter complexes recapitulate nucleosome-like functions?

The general transcription initiation factor TFIID was originally identified, purified, and characterized with a biochemical assay in which accurate transcription initiation is reconstituted with multiple, chromatographically separable activities. Biochemical analyses have demonstrated that TFIID is a multiprotein complex that directs preinitiation complex assembly on both TATA box-containing and TATA-less promoters, and some TFIID subunits have been shown to be molecular targets for activation domains in DNA-binding regulatory proteins. These findings have most commonly been interpreted to support the view that transcriptional activation by upstream factors is the result of enhanced TFIID recruitment to the core promoter. Recent insights into the architecture and cell-cycle regulation of the multiprotein TFIID complex prompt both a reassessment of the functional role of TFIID in gene activation and a review of some of the less well-appreciated literature on TFIID. We present a speculative model for diverse functional roles of TFIID in the cell, explore the merits of the model in the context of published data, and suggest experimental approaches to resolve unanswered questions. Finally, we point out how the proposed functional roles of TFIID in eukaryotic class II transcription fit into a model for promoter recognition and activation that applies to both eubacteria and eukaryotes.

Cloning the human and mouse MMS19 genes and functional complementation of a yeast mms19 deletion mutant

The MMS19 gene of the yeast Saccharomyces cerevisiae encodes a polypeptide of unknown function which is required for both nucleotide excision repair (NER) and RNA polymerase II (RNAP II) transcription. Here we report the molecular cloning of human and mouse orthologs of the yeast MMS19 gene. Both human and Drosophila MMS19 cDNAs correct thermosensitive growth and sensitivity to killing by UV radiation in a yeast mutant deleted for the MMS19 gene, indicating functional conservation between the yeast and mammalian gene products. Alignment of the translated sequences of MMS19 from multiple eukaryotes, including mouse and human, revealed the presence of several conserved regions, including a HEAT repeat domain near the C-terminus. The presence of HEAT repeats, coupled with functional complementation of yeast mutant phenotypes by the orthologous protein from higher eukaryotes, suggests a role of Mms19 protein in the assembly of a multiprotein complex(es) required for NER and RNAP II transcription. Both the mouse and human genes are ubiquitously expressed as multiple transcripts, some of which appear to derive from alternative splicing. The ratio of different transcripts varies in several different tissue types.