Structural, biochemical and functional analyses of tRNA-monooxygenase enzyme MiaE from Pseudomonas putida provide insights into tRNA/MiaE interaction.

MiaE (2-methylthio-N6-isopentenyl-adenosine37-tRNA monooxygenase) is a unique non-heme diiron enzyme that catalyzes the O2-dependent post-transcriptional allylic hydroxylation of a hypermodified nucleotide 2-methylthio-N6-isopentenyl-adenosine (ms2i6A37) at position 37 of selected tRNA molecules to produce 2-methylthio-N6-4-hydroxyisopentenyl-adenosine (ms2io6A37). Here, we report the in vivo activity, biochemical, spectroscopic characterization and X-ray crystal structure of MiaE from Pseudomonas putida. The investigation demonstrates that the putative pp-2188 gene encodes a MiaE enzyme. The structure shows that Pp-MiaE consists of a catalytic diiron(III) domain with a four alpha-helix bundle fold. A docking model of Pp-MiaE in complex with tRNA, combined with site directed mutagenesis and in vivo activity shed light on the importance of an additional linker region for substrate tRNA recognition. Finally, krypton-pressurized Pp-MiaE experiments, revealed the presence of defined O2 site along a conserved hydrophobic tunnel leading to the diiron active center.

Cross-Linked Artificial Enzyme Crystals as Heterogeneous Catalysts for Oxidation Reactions.

Designing systems that merge the advantages of heterogeneous catalysis, enzymology, and molecular catalysis represents the next major goal for sustainable chemistry. Cross-linked enzyme crystals display most of these essential assets (well-designed mesoporous support, protein selectivity, and molecular recognition of substrates). Nevertheless, a lack of reaction diversity, particularly in the field of oxidation, remains a constraint for their increased use in the field. Here, thanks to the design of cross-linked artificial nonheme iron oxygenase crystals, we filled this gap by developing biobased heterogeneous catalysts capable of oxidizing carbon-carbon double bonds. First, reductive O2 activation induces selective oxidative cleavage, revealing the indestructible character of the solid catalyst (at least 30 000 turnover numbers without any loss of activity). Second, the use of 2-electron oxidants allows selective and high-efficiency hydroxychlorination with thousands of turnover numbers. This new technology by far outperforms catalysis using the inorganic complexes alone, or even the artificial enzymes in solution. The combination of easy catalyst synthesis, the improvement of "omic" technologies, and automation of protein crystallization makes this strategy a real opportunity for the future of (bio)catalysis.

The activation of the atypical PKC zeta in light-induced retinal degeneration and its involvement in L-DNase II control.

Light-induced retinal degeneration is characterized by photoreceptor cell death. Many studies showed that photoreceptor demise is caspase-independent. In our laboratory we showed that leucocyte elastase inhibitor/LEI-derived DNase II (LEI/L-DNase II), a caspase-independent apoptotic pathway, is responsible for photoreceptor death. In this work, we investigated the activation of a pro-survival kinase, the protein kinase C (PKC) zeta. We show that light exposure induced PKC zeta activation. PKC zeta interacts with LEI/L-DNase II and controls its DNase activity by impairing its nuclear translocation. These results highlight the role of PKC zeta in retinal physiology and show that this kinase can control caspase-independent pathways.

Apoptosis-inducing factor (AIF) and leukocyte elastase inhibitor/L-DNase II (LEI/LDNaseII), can interact to conduct caspase-independent cell death.

Programmed cell death is an important factor in tissue homeostasis. Lot of work has been performed to characterize the caspase-dependent cell death. Caspase-independent cell death, although important in many physiological situations, is less investigated. In this work we show that two caspase-independent effectors of cell death, namely apoptosis-inducing factor and leukocyte elastase inhibitor derived DNase II interact and can cooperate to induce cell death. These results contribute to the knowledge of molecular pathways of cell death, an important issue in the development of new therapeutic strategies for the treatment of cancer or neurodegenerative diseases.

Conformational modification of serpins transforms leukocyte elastase inhibitor into an endonuclease involved in apoptosis.

The best-characterized biochemical feature of apoptosis is degradation of genomic DNA into oligonucleosomes. The endonuclease responsible for DNA degradation in caspase-dependent apoptosis is caspase-activated DNase. In caspase-independent apoptosis, different endonucleases may be activated according to the cell line and the original insult. Among the known effectors of caspase-independent cell death, L-DNase II (LEI [leukocyte elastase inhibitor]-derived DNase II) has been previously characterized by our laboratory. We have thus shown that this endonuclease derives from the serpin superfamily member LEI by posttranslational modification (A. Torriglia, P. Perani, J. Y. Brossas, E. Chaudun, J. Treton, Y. Courtois, and M. F. Counis, Mol. Cell. Biol. 18:3612-3619, 1998). In this work, we assessed the molecular mechanism involved in the change in the enzymatic activity of this molecule from an antiprotease to an endonuclease. We report that the cleavage of LEI by elastase at its reactive center loop abolishes its antiprotease activity and leads to a conformational modification that exposes an endonuclease active site and a nuclear localization signal. This represents a novel molecular mechanism for a complete functional conversion induced by changing the conformation of a serpin. We also show that this molecular transformation affects cellular fate and that both endonuclease activity and nuclear translocation of L-DNase II are needed to induce cell death.

Nuclear export of LEI/L-DNase II by Crm1 is essential for cell survival.

LEI/L-DNase II is the key protein of a caspase-independent pathway activated by serine proteases. LEI (Leukocyte elastase inhibitor), L-DNase II precursor, is a member of the clade B serpins (also called serpin b1). In its native conformation it inhibits several intracellular proteases and has an anti-apoptotic activity. Following a metabolic stress and the increase of protease activity in the cell, LEI is cleaved and transformed into L-DNase II (LEI-derived DNase II). This transformation is due to a conformational modification that exposes a nuclear localization signal and an endonuclease active site. In this paper we show that LEI can bind the exportin Crm1, and we identify on LEI a nuclear export signal involved in the control of LEI/L-DNase II nuclearization in healthy cells. Point mutation of this site increases the accumulation of the molecule in the nucleus and triggers cell death.

Leukocyte Elastase Inhibitor, the precursor of L-DNase II, inhibits apoptosis by interfering with caspase-8 activation.

LEI (Leukocyte Elastase Inhibitor), the precursor of the pro-apoptotic molecule L-DNase II, belongs to the ovalbumin subgroup of serpins. Several serpins can inhibit apoptosis: the viral serpin Crm A inhibits Fas or TNFalpha-induced apoptosis, and overexpression of PAI-2 or PI-9 protects cells from TNFalpha or granzyme B induced apoptosis. We have previously shown that LEI overexpression protects cells from etoposide-induced apoptosis. The molecular reason of this anti-apoptotic activity is now investigated. We show that, in BHK-21 and HeLa cells, LEI anti-protease activity is essential for its anti-apoptotic effect. The protease inhibited is cathepsin D, released from the lysosome during etoposide treatment. Cathepsin D enhances caspase activity in the cell by cleaving procaspase-8 and LEI overexpression slows down this cleavage, protecting cells from apoptosis. This let us presume that high expression of LEI in tumor cells may reduce the efficiency of etoposide as a chemotherapeutic agent.

Ruminococcin C, a promising antibiotic produced by a human gut symbiont.

A major public health challenge today is the resurgence of microbial infections caused by multidrug-resistant strains. Consequently, novel antimicrobial molecules are actively sought for development. In this context, the human gut microbiome is an under-explored potential trove of valuable natural molecules, such as the ribosomally-synthesized and post-translationally modified peptides (RiPPs). The biological activity of the sactipeptide subclass of RiPPs remains under-characterized. Here, we characterize an antimicrobial sactipeptide, Ruminococcin C1, purified from the caecal contents of rats mono-associated with Ruminococcus gnavus E1, a human symbiont. Its heterologous expression and post-translational maturation involving a specific sactisynthase establish a thioether network, which creates a double-hairpin folding. This original structure confers activity against pathogenic Clostridia and multidrug-resistant strains but no toxicity towards eukaryotic cells. Therefore, the Ruminococcin C1 should be considered as a valuable candidate for drug development and its producer strain R. gnavus E1 as a relevant probiotic for gut health enhancement.

LEI/L-DNase II: interplay between caspase-dependent and independent pathways.

Caspase activation has been seen, for several years, as the biochemical marker of apoptosis. However, in 2005 the Nomenclature Committee on Cell Death (NCCD) established that the 'official' classification of cell death had to rely on morphological criteria owing to the absence of a clear-cut equivalence between structural alterations and biochemical pathways. Actually, the controlled destruction of the cell is coordinated by a proteolytic system involving caspases but also other proteases like cathepsins, calpains and serine proteases. These enzymes participate in an activation cascade that culminates in cleavage of a set of proteins resulting in disassembly of the cell. This disassembling also includes the activation of endonucleases that will destroy a potentially harmful DNA. A caspase-activated DNase performs DNA degradation in caspase-dependent apoptosis, but other endonucleases like L-DNase II or GAAD are activated in caspase-independent apoptosis, allowing the complete dismantling of the cell.

Leukocyte elastase inhibitor: a new regulator of PARP-1.

Poly(ADP-ribose) polymerase-1 (PARP-1) uses NAD(+) as a substrate to form ADP-ribose. During apoptosis, caspases cleave PARP-1 to avoid excessive NAD consumption. Because PARP-1 is a key regulator of the activity of DNases involved in caspase-dependent apoptosis, its cleavage is required to promote DNA degradation. To explore the situation in caspase-independent cell death, we investigated the effect of PARP-1 on the acid endonuclease leukocyte elastase inhibitor (LEI)-derived DNase II (L-DNase II). We found for the first time an association between PARP-1 and LEI/L-DNase II. Unexpectedly, we observed that LEI influenced the automodification of PARP-1.

Molecular mechanism of L-DNase II activation and function as a molecular switch in apoptosis.

The discovery of caspase activation counts as one of the most important finds in the biochemistry of apoptosis. However, targeted disruption of caspases does not impair every type of apoptosis. Other proteases can replace caspases and several so called "caspase independent" pathways are now described. Here we review our current knowledge on one of these pathways, the LEI/L-DNase II. It is a serine protease-dependent pathway and its key event is the transformation of LEI (leukocyte elastase inhibitor, a serine protease inhibitor) into L-DNase II (an endonuclease). The molecular events leading to this change of enzymatic function as well as the cross-talk and interactions of this molecule with other apoptotic pathway, including caspases, are discussed.