Jean-François Dierick

Proteome analysis and identification of symbiosis‐related proteins from Medicago truncatula Gaertn. by two‐dimensional electrophoresis and mass spectrometry

Time‐course analysis of root protein profiles was studied by two‐dimensional gel electrophoresis and silver staining in the model plant Medicago truncatula, inoculated either with the arbuscular mycorrhizal fungus Glomus mosseae or with the nitrogen fixing bacterium Sinorhizobium meliloti. Protein modifications in relation to the development of both symbioses included down‐ and upregulations, as well as newly induced polypeptides. Matrix assisted laser desorption/ionization‐time of flight‐mass spectrometry after trypsin digestion clearly identified one polypeptide induced in nodulated roots as a M. truncatula leghemoglobin. Internal sequencing with a quadrupole time‐of‐flight mass spectrometer and database searches confirmed the induction of proteins previously described in root symbioses, and revealed the implication of other proteins. In nodulated roots, one polypeptide was identified as an elongation factor Tu from S. meliloti, while another one could not be assigned a function. In mycorrhizal roots, analyzed proteins also included a protein of unknown function, as well as a glutathione‐S‐transferase, a fucosidase, a myosin‐like protein, a serine hydroxymethyltransferase and a cytochrome‐c‐oxidase. These results emphasize the usefulness of proteome analysis in identifying molecular events occurring in plant root symbioses.

Stress-induced premature senescence. Essence of life, evolution, stress, and aging.

The stress syndrome was discovered accidentally by Hans Selye while searching for new hormones in the placenta.1 After injecting rats with crude preparations, Selye found adrenal enlargements and involution of thymus and lymph nodes, which he thought were specific for a particular hormone. It occurred to Selye that these symptoms might represent a nonspecific response to noxious agents. Indeed, this was found to be the case when he injected rats with diverse agents. Selye defined the stress response as the “general adaptation syndrome.”2,3 According to this theory, the initial reaction to stress is shock, it is followed by a countershock phase, and gradually resistance develops to the stressor. This resistance may turn into exhaustion, however, if the stressor persists, and death may ensue. Both specific and nonspecific resistance develops during stress.4 In his last scientific book, Selye defined biologic stress as “the non-specific response of the body to any demand made upon it.”5 Beside the transfer of the word “stress” from physics to biology, Selye also coined the words corticosteroids, glucocorticoids, and mineralocorticoids.6 Nowadays, the concept of stress has invaded most fields of the biologic, medical, and social sciences. Cellular and molecular biology has become interested in the study of the stress response of human, animal, and plant cells, the consensus being that “any environmental factor potentially unfavourable to living organism” is stress.7 It is also generally agreed that “if the limits of tolerance are exceeded and the adaptive capacity is over-worked, the result may be permanent damage or even death.”8 Three phases of the stress response have been defined based on experimental observations: (1) the response phase of alarm reaction with deviation of functional norm, decline of vitality, and excess of catabolic processes over anabolism, (2) the restitution phase or stage of resistance with adaptation processes and repair processes, and (3) either the end phase, that stage of exhaustion or long-term response when stress intensity is too high, leading to overcharge of the adaptation capacity, damage, chronic dis-

Identification of 30 protein species involved in replicative senescence and stress‐induced premature senescence

Exposure of human proliferative cells to subcytotoxic stress triggers stress‐induced premature senescence (SIPS) which is characterized by many biomarkers of replicative senescence. Proteomic comparison of replicative senescence and stress‐induced premature senescence indicates that, at the level of protein expression, stress‐induced premature senescence and replicative senescence are different phenotypes sharing however similarities. In this study, we identified 30 proteins showing changes of expression level specific or common to replicative senescence and/or stress‐induced premature senescence. These changes affect different cell functions, including energy metabolism, defense systems, maintenance of the redox potential, cell morphology and transduction pathways.

Stress-Induced Premature Senescence or Stress-Induced Senescence-Like Phenotype: One In Vivo Reality, Two Possible Definitions?

No consensus exists so far on the definition of cellular senescence. The narrowest definition of senescence is irreversible growth arrest triggered by telomere shortening counting cell generations (definition 1). Other authors gave an enlarged functional definition encompassing any kind of irreversible arrest of proliferative cell types induced by damaging agents or cell cycle deregulations after overexpression of proto-oncogenes (definition 2). As stress increases, the proportion of cells in “stress-induced premature senescence-like phenotype” according to definition 1 or “stress-induced premature senescence,” according to definition 2, should increase when a culture reaches growth arrest, and the proportion of cells that reached telomere-dependent replicative senescence due to the end-replication problem should decrease. Stress-induced premature senescence-like phenotype and telomere-dependent replicatively senescent cells share basic similarities such as irreversible growth arrest and resistance to apoptosis, which may appear through different pathways. Irreversible growth arrest after exposure to oxidative stress and generation of DNA damage could be as efficient in avoiding immortalisation as “telomere-dependent” replicative senescence. Probabilities are higher that the senescent cells (according to definition 2) appearing in vivo are in stress-induced premature senescence rather than in telomere-dependent replicative senescence. Examples are given suggesting these cells affect in vivo tissue (patho)physiology and aging.

Growth kinetics rather than stress accelerate telomere shortening in cultures of human diploid fibroblasts in oxidative stress-induced premature senescence

WI‐38 human diploid fibroblasts underwent accelerated telomere shortening (490 bp/stress) and growth arrest after exposure to four subcytotoxic 100 μM tert‐butylhydroperoxide (t‐BHP) stresses, with a stress at every two population doublings (PD). After subcytotoxic 160 μM H2O2 stress or five repeated 30 μM t‐BHP stresses along the same PD, respectively a 322±55 and 380±129 bp telomere shortening was observed only during the first PD after stress. The percentage of cells resuming proliferation after stress suggests this telomere shortening is due to the number of cell divisions accomplished to reach confluence during the first PD after stress.

Stress-induced premature senescence as alternative toxicological method for testing the long-term effects of molecules under development in the industry.

No alternative in vitro method exists fordetecting the potential long-term genotoxic effects ofmolecules at subcytotoxic concentrations, in terms ofdays and weeks after exposure(s) to the moleculetested. A theoretical model of cellular senescence ledto the concept that subcytotoxic stresses under anymolecules at subcytotoxic doses, such as moleculesunder development in the pharmaceutical, cosmetics andfood industry, might lead human fibroblasts into a stateclosely related to in vitro senescence. Thisconcept was then experimentally confirmed invitro: many biomarkers of replicative senescence ofhuman fibroblasts were found 72 h after theirexposure to various kinds of stressors used at non-cytotoxic concentrations. This phenomenon has beentermed stress-induced premature senescence (SIPS).Moreover, proteomics studies have revealed that,besides their effects on the appearance of thebiomarkers of senescence, sublethal stresses under avariety of stressors also lead to long-term specificchanges in the expression level of proteins which arestress-specific. These changes have been coined themolecular scars of stress. The proteins correspondingto these molecular scars may be identified using thelatest developments in mass spectrometry. This modelof stress-induced premature senescence may be appliedto the toxicological sciences when testing for thepotential irreversible long-term effects of moleculeson the cell fate.

Proteomics in experimental gerontology

The first gerontological studies using two-dimensional gel electrophoresis (2DGE) were frustrating since it was very difficult, when not impossible, to identify the proteins for which an age-related change in expression level was suspected. Reproducibility was also a main pitfall. Accumulated progress in 2DGE and especially the development of mass spectrometry of proteins and peptides gave accessibility to the routine identification of differentially expressed proteins. A new paradigm was born: proteomics. In addition to expression changes, post-translational modifications are included in proteomics, and will be more and more studied using mass spectrometry. After a review of the current developments of 2DGE and mass spectrometry, we shall discuss how the technologies currently available in proteomics could give fresh impetus to experimental gerontology, complementary to more recent approaches based on wide expression analysis tools such as DNA and protein arrays.

Role of TGF-β1-independent changes in protein neosynthesis, p38αMAPK, and cdc42 in hydrogen peroxide-induced senescence-like morphogenesis

The role of TGF-beta1 in hydrogen peroxide-induced senescence-like morphogenesis has been described. The aim of this work was to investigate whether TGF-beta1-independent changes in protein synthesis are involved in this morphogenesis and to study possible mechanisms occurring earlier than TGF-beta1 overexpression. Among the multiple TGF-beta1-independent changes in protein neosynthesis, followed or not by posttranslational modifications, identified by proteomic analysis herein, those of ezrin, L-caldesmon, and HSP27 were particularly studied. Rho-GTPase cdc42 was shown to be responsible for p38(MAPK) activation, in turn triggering phosphorylation of L-caldesmon and HSP27. Cdc42 was also shown to be mainly responsible for the increase in TGF-beta1 mRNA level observed at 24 h after treatment with H(2)O(2) and onward. This study further clarified the mechanisms of senescence-like morphogenesis in addition to the previously demonstrated role of TGF-beta1 signaling pathways.

Hormesis: a quest for virtuality?

The birth of new concepts brings constructive confrontations with older concepts. The use of the concept of hormesis in biogerontology is legitimate since the role of stress is fundamental in both aging and hormesis. Are there theoretical restrictions against a generalised role of hormesis in aging? What are the possible side effects? Can hormesis be reconciliated with the life span-prolonging effects of caloric restriction? The aim of this short paper is to propose clues to answer these important questions.

Transcriptome and Proteome Analysis in Human Senescent Fibroblasts and Fibroblasts Undergoing Premature Senescence Induced by Repeated Sublethal Stresses

JEAN-FRANÇOIS DIERICK,a,b,c,d,e THIERRY PASCAL,b,e FLORENCE CHAINIAUX,b FRANÇOIS ELIAERS,b JOSÉ REMACLE,b PETER MOSE LARSEN,c PETER ROEPSTORFF,d AND OLIVIER TOUSSAINTa,b bDepartment of Biology, Unit of Cellular Biochemistry and Biology, University of Namur (FUNDP), 5000 Namur, Belgium cThe Centre for Proteome Analysis in Life Sciences, International Science Park Odense, 5230 Odense M, Denmark dDepartment of Molecular Biology, University of Southern Denmark, 5230 Odense M, Denmark