Antonio Jiménez-Ruiz

Apoptotic markers in protozoan parasites

The execution of the apoptotic death program in metazoans is characterized by a sequence of morphological and biochemical changes that include cell shrinkage, presentation of phosphatidylserine at the cell surface, mitochondrial alterations, chromatin condensation, nuclear fragmentation, membrane blebbing and the formation of apoptotic bodies. Methodologies for measuring apoptosis are based on these markers. Except for membrane blebbing and formation of apoptotic bodies, all other events have been observed in most protozoan parasites undergoing cell death. However, while techniques exist to detect these markers, they are often optimised for metazoan cells and therefore may not pick up subtle differences between the events occurring in unicellular organisms and multi-cellular organisms.In this review we discuss the markers most frequently used to analyze cell death in protozoan parasites, paying special attention to changes in cell morphology, mitochondrial activity, chromatin structure and plasma membrane structure/permeability. Regarding classical regulators/executors of apoptosis, we have reviewed the present knowledge of caspase-like and nuclease activities.

A Leucine-Rich Repeat Motif of Leishmania Parasite Surface Antigen 2 Binds to Macrophages through the Complement Receptor 3

Membrane glycoconjugates on the Leishmania parasites, notably leishmanolysin and lipophosphoglycan, have been implicated in attachment and invasion of host macrophages. However, the function of parasite surface Ag 2 (PSA-2) and membrane proteophosphoglycan (PPG) has not been elucidated. In this study we demonstrate that native and recombinant Leishmania infantum PSA-2, which consists predominantly of 15 leucine-rich repeats (LRR) and a recombinant LRR domain derived from L. major PPG, bind to macrophages. The interaction is restricted to macrophages and appears to be calcium independent. We have investigated the PSA-2-macrophage interaction to identify the host receptor involved in binding and we show that binding of PSA-2 to macrophages can be blocked by Abs to the complement receptor 3 (CR3, Mac-1). Data derived from mouse macrophage studies were further confirmed using cell lines expressing human CR3, and showed that PSA-2 also binds to the human receptor. This is the first demonstration of a functional role for PSA-2. Our data indicate that in addition to leishmanolysin and lipophosphoglycan, parasite attachment and invasion of macrophages involve a third ligand comprising the LRRs shared by PSA-2 and PPG and that these interactions occur via the CR3.

Leishmania infantum expresses a mitochondrial nuclease homologous to EndoG that migrates to the nucleus in response to an apoptotic stimulus

It is increasingly accepted that single-celled organisms, such as Leishmania parasites, are able to undergo a cell death process that resembles apoptosis in metazoans and is induced by a variety of stimuli. However, the molecular mechanisms that participate and regulate this death process are still very poorly described, and very few of the participating molecules have been identified. Because DNA degradation is probably the most frequently characterized event during programmed cell death in Leishmania parasites, we have focused on identifying a candidate nuclease responsible for this effect during the cell death process. The results presented herein demonstrate that Leishmania infantum promastigotes express a nuclease similar to the endonuclease G of higher eukaryotes which, according to its predicted structure, belongs to the beta beta alpha metal superfamily of nucleases. Its cation dependence resembles that of the EndoGs present in other organisms and, similarly to them, it is inhibited by moderate concentrations of K+ or Na+. L. infantum EndoG contains a signal peptide that causes its translocation to the mitochondrion where it is maintained under normal growth conditions. However, under the pressure of a death stimulus such as edelfosine treatment, L. infantum EndoG is released from the single mitochondrion and translocates to the nucleus, where it is thought to participate in the process of DNA degradation that is associated with programmed cell death. Our results also demonstrate that overexpression of the nuclease in edelfosine-treated promastigotes causes a significant increase in the percentage of TUNEL-positive parasites.

Selection of aptamers against KMP-11 using colloidal gold during the SELEX process

SELEX procedure is a methodology in which single stranded oligonucleotides are selected from a wide variety of sequences based on their interaction with a target molecule. We have designed a novel SELEX methodology using colloidal gold to select high affinity single stranded DNA aptamers against Leishmania infantum KMP-11. Kinetoplastid membrane protein-11 (KMP-11) is a major component of the cell membrane of kinetoplastid parasites. Although its function is not known, the fact that KMP-11 is a cytoskeleton-associated protein suggests that it may be involved in mobility or in some other aspects of the flagellar structure. We have isolated a single stranded DNA aptamer population that binds specifically to L. infantum KMP-11. This population has been characterized in a series of in vitro experiments suggesting that it may be used as a powerful tool to further investigate the role of KMP-11 during Leishmania development and/or as a diagnostic tool in Leishmania infection.

The expression of the Leishmania infantum KMP-11 protein is developmentally regulated and stage specific.

Transcription of the gene coding for the KMP-11 protein of Leishmania infantum results in the production of a mature RNA transcript of 1.3 kb in length. The expression of KMP-11 mRNA is strongly down-regulated not only during the parasite growth from the logarithmic to the stationary phase but also during the differentiation transit from promastigotes to amastigotes. The estimated concentration of KMP-11 is one order of magnitude higher in promastigotes than in amastigotes. The analysis of the Triton X-114 phase partition of the protein shows that, in agreement with its predicted secondary structure, KMP-11 has an amphipathic nature since it is found in the aqueous as well as in the detergent phase. By fluorescence microscopy a defined pattern of distribution of the protein was observed only in promastigotes where KMP-11 is mainly located in the flagellum and the flagellar pocket.

Edelfosine Induces an Apoptotic Process in Leishmania infantum That Is Regulated by the Ectopic Expression of Bcl-XL and Hrk

ABSTRACT The alkyl-lysophospholipids edelfosine and miltefosine induce apoptosis in Leishmania infantum promastigotes. The finding that edelfosine-induced cell death can be regulated by the ectopic expression of the antiapoptotic and proapoptotic members of the Bcl-2 family of proteins Bcl-XL and Hrk suggests that this process is similar to apoptosis in eukaryotic cells.

Guidelines and recommendations on yeast cell death nomenclature

Elucidating the biology of yeast in its full complexity has major implications for science, medicine and industry. One of the most critical processes determining yeast life and physiology is cellular demise. However, the investigation of yeast cell death is a relatively young field, and a widely accepted set of concepts and terms is still missing. Here, we propose unified criteria for the definition of accidental, regulated, and programmed forms of cell death in yeast based on a series of morphological and biochemical criteria. Specifically, we provide consensus guidelines on the differential definition of terms including apoptosis, regulated necrosis, and autophagic cell death, as we refer to additional cell death routines that are relevant for the biology of (at least some species of) yeast. As this area of investigation advances rapidly, changes and extensions to this set of recommendations will be implemented in the years to come. Nonetheless, we strongly encourage the authors, reviewers and editors of scientific articles to adopt these collective standards in order to establish an accurate framework for yeast cell death research and, ultimately, to accelerate the progress of this vibrant field of research.

Protein-protein interactions at an enzyme-substrate interface: characterization of transient reaction intermediates throughout a full catalytic cycle of Escherichia coli thioredoxin reductase.

A large collection of structural snapshots along a full catalytic cycle of Escherichia coli thioredoxin reductase (TrxR) has been generated and characterized using a combination of theoretical methods. Molecular models were built starting from the available X‐ray crystallographic structures of dimeric wild‐type TrxR in the flavin‐oxidizing conformation and a C135S TrxR mutant enzyme in a flavin‐reducing conformation “trapped” by a cross‐link between Cys138 of TrxR and Cys32 of C35S mutant thioredoxin (Trx). The transition between these two extreme states, which is shown to be reproduced in a normal mode analysis, as well as natural cofactor binding and dissociation, were simulated for the wild‐type species using unrestrained and targeted molecular dynamics following docking of oxidized Trx to reduced TrxR. The whole set of simulations provides a comprehensive structural framework for understanding the mechanism of disulfide reduction in atomic detail and identifying the most likely intermediates that facilitate entry of NADPH and exit of NADP+. The crucial role assigned to Arg73 and Lys36 of Trx in substrate binding and complex stabilization was ascertained when R73G, R73D, and K36A site‐directed mutants of Trx were shown to be impaired to different extents in their ability to be reduced by TrxR. On the basis of previous findings and the results reported herein, E. coli TrxR appears as a beautifully engineered molecular machine that is capable of synchronizing cofactor capture and ejection with substrate binding and redox activity through an interdomain twisting motion. Proteins 2010.

Novel hybrid selenosulfonamides as potent antileishmanial agents.

Diselenide and sulfonamide derivatives have recently attracted considerable interest as leishmanicidal agents in drug discovery. In this study, a novel series of sixteen hybrid selenosulfonamides has been synthesized and screened for their in vitro activity against Leishmania infantum intracellular amastigotes and THP-1 cells. These assays revealed that most of the compounds exhibited antileishmanial activity in the low micromolar range and led us to identify three lead compounds (derivatives 2, 7 and 14) with IC50 values ranging from 0.83 to 1.47 μM and selectivity indexes (SI) over 17, much higher than those observed for the reference drugs miltefosine and edelfosine. When evaluated against intracellular amastigotes, hybrid compound 7 emerged as the most active compound (IC50 = 2.8 μM), showing higher activity and much less toxicity against THP-1 cells than edelfosine. These compounds could potentially serve as templates for future drug-optimization and drug-development efforts for their use as therapeutic agents in developing countries.

Novel heteroaryl selenocyanates and diselenides as potent antileishmanial agents

ABSTRACT A series of new selenocyanates and diselenides bearing interesting bioactive scaffolds (quinoline, quinoxaline, acridine, chromene, furane, isosazole, etc.) was synthesized, and their in vitro leishmanicidal activities against Leishmania infantum amastigotes along with their cytotoxicities in human THP-1 cells were determined. Interestingly, most tested compounds were active in the low micromolar range and led us to identify four lead compounds (1h, 2d, 2e, and 2f) with 50% effective dose (ED50) values ranging from 0.45 to 1.27 μM and selectivity indexes of >25 for all of them, much higher than those observed for the reference drugs. These active derivatives were evaluated against infected macrophages, and in order to gain preliminary knowledge about their possible mechanism of action, the inhibition of trypanothione reductase (TryR) was measured. Among these novel structures, compounds 1h (3,5-dimethyl-4-isoxazolyl selenocyanate) and 2d [3,3′-(diselenodiyldimethanediyl)bis(2-bromothiophene)] exhibited good association between TryR inhibitory activity and antileishmanial potency, pointing to 1h, for its excellent theoretical ADME (absorption, distribution, metabolism, and excretion) properties, as the most promising lead molecule for leishmancidal drug design.