WO2006130190A2 - Proline suppresses apoptosis - Google Patents

Abstract

The discovery that proline is a potent antioxidant and inhibitor of programmed cell death is described, as are proline-containing compositions and methods for using such compositions in response to, or in advance of, experiencing oxidative stress. These compositions and methods find application across the animal and plant kingdoms. Transgenic plants exhibiting altered intracellular proline levels, as compared to plants lacking such an alteration, are also described, as are transgenic plants that exhibit altered expression of one or more genes involved in the metabolism of reactive oxygen species (ROS), as compared to unmodified plants.

Description

Proline Suppresses Apoptosis

GOVERNMENT INTEREST

At least some of the subject matter described in this specification resulted from the use of U.S. government funds (National Science Foundation Grant IBN 0133078). As a result, the U.S. government may have certain rights in these inventions.

RELATED APPLICATION

This application claims the benefit of, and priority to, the following U.S. provisional patent application: serial number 60/651,571, filed 7 February 2005 and entitled, "Proline suppresses apoptosis in the fungal pathogen Colletotrichum tήfolii", which application is incorporated herein by reference in its entirety, including figures and claims, for any and all purposes.

FIELD OF THE INVENTION

This invention relates generally to modulating stress resistance in eukaryotic cells and organisms. In particular, it has been found that the amino acid proline can modulate stress resistance in plants and animals, including cells in culture, due to its previously unknown capacity to reduce intracellular levels of reactive oxygen species (ROS). Accordingly, proline can be applied, for example, to plants, cells in culture, etc. so as to confer resistance, or tolerance, to stress, as compared to untreated plants.

BACKGROUND OF THE INVENTION

1. Introduction.

The following description includes information that may be useful in understanding the present invention. It is not an admission that any such information is prior art, or relevant, to the presently claimed inventions, or that any publication specifically or implicitly referenced is prior art.

2. Background.

The global population is predicted to exceed 7 billion in the next ten years. Population growth places extreme pressure on the world food supply and thus ability to increase agricultural productivity is of utmost importance. The development of strategies to mitigate crop loss caused by vast of array on insults has far reaching potential in increasing agricultural performance.

Programmed cell death ("PCD") and its morphological equivalent, apoptosis, is tightly regulated process by which a eukaryotic cell undergoes suicide. PCD has been found to be an intrinsic part of the development, maintenance of cellular homeostasis, and defense against environmental insults, including pathogen attack, in animals. Plants often demonstrate PCD-like phenotypes following a numerous biotic and abiotic stresses. As a result of many insults (stresses) to a plant, reactive oxygen species (ROS) accumulate, usually as a direct result of unregulated or dysfunctional photon-driven electron transport, a major component of the photosynthetic process. ROS has been established to maintain important roles in cellular signaling pathways including: cell communication, control of gene expression, and oxygen sensing. Contrarily, ROS has been demonstrated to be damaging to the cell. The cytotoxic activity of ROS can be attributed to its unstable and highly reactive nature, which can damage biomolecules including proteins, nucleic acids and especially lipids. Inappropriate regulation of ROS levels can lead to diseased states. Therefore, reducing the levels of ROS that are generated both during normal physiological and numerous pathological states of plants, could curtail the associated PCD and confer stress resistance or a growth advantage to the plant.

Proline is an amino acid that, in addition to being a component of proteins, is known to be utilized as an osmolyte in bacteria, mammals and plants. It is not, however, known to have anti-oxidant, particularly ROS-scavenging, activity. As described herein, the previously unappreciated ROS-scavenging property of proline can be used to prevent or reverse the induction of programmed cell death by ROS generated during stress, including biotic and abiotic stresses such as nutritional stress, UV light, cold, heat, high salt, and hydrogen peroxide exposure.

3. Definitions.

Before describing the instant invention in detail, several terms used in the context of the present invention will be defined. In addition to these terms, others are defined elsewhere in the specification, as necessary. Unless otherwise expressly defined herein, terms of art used in this specification will have their art-recognized meanings.

An "abiotic" insult or stress refers to a plant challenge caused by exposure to a non- viable or non-living agent {i.e., an abiotic agent). Examples of abiotic agents that can cause an abiotic stress include environmental factors such as low moisture (drought), high moisture (flooding), nutrient deficiency, radiation levels, air pollution (ozone, acid rain, sulfur dioxide, etc.), high temperature (hot extremes or heat shock), low temperature (cold extremes or cold shock), and soil toxicity (e.g., toxic levels of salt, heavy metals, etc.), as well as herbicide damage, pesticide damage, or other agricultural practices (e.g., over-fertilization, improper use of chemical sprays, etc.).

The term "acceptable salt" refers to any salt of an active ingredient (e.g., proline) in which the active ingredient retains its biological effectiveness following administration. Salts include acid and base salts. In the context of human administration, the salt is preferably a salt that is pharmaceutically acceptable, i.e., is a "pharmaceutically acceptable salt". Acceptable acid addition salts may be prepared from inorganic and organic acids, while acceptable base addition salts can be prepared from inorganic and organic bases. For a review of pharmaceutically acceptable salts, see Berge, et a ((1977) J. Pharm. ScL, vol. 66, 1).

The terms "administration", "administering", "delivery", and the like refer to any administration or delivery of a composition according to the invention for the purpose of treating or preventing an effect caused by, or correlated with, a stress.

A "biotic" insult or stress refers to a plant challenge caused by viable or biologic agents (i.e., biotic agents). Examples of biotic agents that can cause a biotic stress include insects, fungi, bacteria, viruses, nematodes, viroids, mycloplasmas, etc.

An "effective amount" refers to an amount of an active ingredient (e.g., proline) sufficient to effect stress reduction when administered to an organism (e.g., and animal or plant) then or subsequently exposed to an environmental stress. In the context of agriculture, an "effective amount" of proline, for example, is one that produces an objectively detectable or measurable change in one or more parameters associated with plant cell survival, including a delay or absence in the appearance of events correlated with apoptosis (e.g., DNA laddering, TUNEL- positive staining, etc.), continued viability, an effective reduction in intracellular ROS levels (i.e., a reduction in ROS levels that is correlated with resistance to, or tolerance of, an environmental stress) etc. Similarly, in the context of animal treatment, an "effective amount" may be referred to as a "therapeutically effective amount", and thus refers to an amount of proline that produces an objectively detectable or measurable change in one or more parameters associated with cell survival, including a delay or absence in the appearance of events correlated with apoptosis. Of course, what constitutes an "effective amount" of proline will vary depending upon the particular organism, its, size, weight, and age, the type and severity of the stress to be countered, the particular compound chosen, the dosing regimen to be followed, the timing of administration, the manner of administration and the like, all of which can readily be determined by one of ordinary skill in the art. It will be appreciated that in the context of combination therapy, what constitutes an "effective amount" of a particular active ingredient may differ from what constitutes an effective amount of the active ingredient when administered as the sole active ingredient (i.e., a treatment regimen that employs only one chemical entity as the active ingredient intended to counter the particular stress(es)).

An "environmental stress" refers to an abiotic or biotic stress.

A "host cell" refers to a cell that contains a vector according to the invention.

The terms "include", "including", and the like mean "including, without limitation".

The term "modulate" refers to the ability to alter from a basal level. As used in the context of apoptosis (e.g., to "modulate" apoptosis or PCD), "modulate" refers to the ability to alter or change any biochemical, physiological, or morphological event associated with apoptosis from its basal level. For example, apoptosis has been "modulated" if there has been an alteration in expression of a gene involved in an apoptotic pathway, the interaction of an apoptotic pathway protein with other proteins, the formation of apoptotic bodies, or the DNA cleavage is altered from its original state. Similarly, response to a stress has been "modulated" if, for example, a biochemical, physiological, or morphological parameter (e.g. , growth, viability, fruit or send production, photosynthetic rate, rate of respiration or transpiration, etc.) being assessed differs from the level of that parameter in the absence of the stress.

"Monotherapy" or "monotreatment" refers to a treatment regimen based on the delivery of one active ingredient, whether administered as a single dose or several doses over time. On the other hand, "combination therapy" or "combination treatment" refers to a treatment regimen that involves the provision of at least two distinct treatments to achieve an indicated effect. For example, a combination therapy may involve the administration of two or more chemically distinct active ingredients, for example, proline and a fertilizer. Alternatively, a combination therapy may involve proline administration as well as the expression of one or more transgenes the effect of which is to counter an effect attributable to reactive oxygen species correlated with occurrence of a stress. In the context of a combination treatment, it is understood that the active ingredients may be administered as part of the same composition, as different compositions, or, in the context of one or more exogenously administered active ingredients and the expression of one or more transgenes in a transgenic plant, as an administered active ingredient (e.g., proline) and a transgene expressed at least one cell type or tissue in the plant being treated. When administered in combination, the different active ingredients may be administered (or expressed, as the case may be) at the same or different times, by the same or different routes, using the same of different dosing regimens, all as the particular context requires and as determined by the party administering at least one of the exogenously delivered active ingredients, for example, a farmer in the context of agriculture and an attending physician or veterinarian in the context of human or animal treatment.

An "organism" refers to any plant or animal that may be treated in accordance with the invention. A subset of such organisms includes patients, where a "patient" refers to an animal in need of treatment that can be effected by molecules of the invention. Animals that can be treated in accordance with the invention include vertebrates, with mammals such as bovine, canine, equine, feline, ovine, porcine, and primate (including humans and non-humans primates) animals being particularly preferred examples.

"Oxidative stress" is intended to refer to cell damage due the generation of free radicals. Oxidative stresses include ionizing radiation, ultraviolet radiation, and reactive oxygen species.

A "patentable" composition, process, machine, or article of manufacture according to the invention means that the subject matter satisfies all statutory requirements for patentability at the time the analysis is performed. For example, with regard to novelty, non-obviousness, or the like, if later investigation reveals that one or more claims encompass one or more embodiments that would negate novelty, non-obviousness, etc., the claim(s), being limited by definition to "patentable" embodiments, specifically exclude the unpatentable embodiment(s). Also, the claims appended hereto are to be interpreted both to provide the broadest reasonable scope, as well as to preserve their validity. Furthermore, if one or more of the statutory requirements for patentability are amended or if the standards change for assessing whether a particular statutory requirement for patentability is satisfied from the time this application is filed or issues as a patent to a time the validity of one or more of the appended claims is questioned, the claims are to be interpreted in a way that (1) preserves their validity and (2) provides the broadest reasonable inteipretation under the circumstances. A "plant" refers to a whole plant, including a plantlet. Suitable plants for use in the invention include any plant amenable to techniques that result in the introduction of nucleic acid into a plant cell, including both dicotyledonous and monocotyledonous plants. Representative examples of dicotyledonous plants include tomato, potato, arabidopsis, tobacco, cotton, rapeseed, field beans, soybeans, peppers, lettuce, peas, alfalfa, clover, cole crops or Brassica oleracea (e.g., cabbage, broccoli, cauliflower, and Brussels sprouts), radish, carrot, beets, eggplant, spinach, cucumber, squash, melons, cantaloupe, sunflowers, and various ornamentals. Representative examples of monocotyledonous plants include asparagus, field and sweet corn, barley, wheat, rice, sorghum, onion, pearl millet, rye and oat, and ornamentals.

The term "plant cell" refers to a cell from, or derived from, a plant, including gamete- producing cells and cells (e.g., protoplasts) which are capable of regenerating into whole plants. When a cell has been transformed with a nucleic acid or vector according to the invention, it is host cell.

The term "plant tissue" includes differentiated and undifferentiated tissues of a plant, including roots, stems, shoots, leaves, pollen, seeds, tumor tissue, and various forms of cells in culture, including cell suspensions, protoplasts, embryos, and callus tissue.

A "plurality" means more than one.

"ROS" refers to reactive oxygen species as defined by an Oxygen atom that is partially reduced having an unpaired electron. This oxygen can be part of a molecule, as found in hydrogen peroxide; an ion such as is the hypochlorite ion; a radical like the hydroxyl radical, or both an ion and a radical, such as the superoxide anion.

The term "species" is used herein in various contexts, e.g., a particular species of a stress- reducing agent. In each context, the term refers to a population of chemically indistinct molecules of the sort referred in the particular context.

The term "transgene" or "heterologous nucleic acid molecule" refers to a nucleic acid molecule containing at least one of a structural gene, a regulatory element (e.g., a promoter, enhancer, etc.). A heterologous nucleic acid molecule generally, although not necessarily, is a nucleic acid molecule isolated from another species. As will be appreciated, the term "transgene" includes a nucleic acid molecule from the same species, where such molecule has been modified or been placed in operable association with on or more regulatory elements (e.g., a promoter) that differs from the natural or wild-type promoter with which the gene is associated in nature.

The terms "treatment", "treating", and the like refer to any treatment of a stress, or consequence of experiencing a stress, including preventing or protecting against the stress, or consequence thereof (for example, causing the clinical symptoms not to develop); inhibiting a consequence of experiencing a stress {i.e., arresting or suppressing the development of the consequence(s)); and/or relieving one or more consequences of experiencing a stress (i.e., causing a halt or reversal of the negative consequence(s) resulting from experiencing the stress). Relatedly, the term "prophylaxis" will be understood to constitute a type of "treatment" that encompasses both "preventing" and "suppressing" the effects of a stress. The term "protection" thus includes "prophylaxis".

An "unmodified" organism or cell refers to a cell or organism that does not contain the particular modification noted. For example, in the context of transgenic plant tissue that contains an engineered genetic modification that results in a different level of catalase gene expression in at least some of the cells of that tissue as compared to unmodified cells, "unmodified" means that those cells do not contain the modification that gave rise to the altered level of catalase gene expression, although this is not to say that such cells do not contain other engineered genetic modifications.

A "wild-type"-plant or plant variety refers to a plant that does not contain a transgene or nucleic acid according to the invention. As such, the plant may, in fact, be a transgenic plant, although any transgene(s) contained in such "wild-type" plant will comprise a nucleic acid other than a nucleic acid according to this invention.

SUMMARY OF THE INVENTION

The role of reactive oxygen species (ROS) in cell communication, control of gene expression, and oxygen sensing is well established. Inappropriate regulation of ROS levels can damage cells, resulting in a diseased state. In Colletotrichum trifolii, a fungal pathogen of alfalfa, the mutationally activated oncogenic fungal Ras (DARas) elevates levels of ROS, causing abnormal fungal growth and development and eventual apoptotic-like cell death, but only when grown under nutrient-limiting conditions. Remarkably, restoration to the wild-type phenotype requires only proline. Thus, this specification describes a previously unrecognized function of proline, namely, its ability to function as a potent antioxidant and inhibitor of programmed cell death, in addition to its well-established role as an osmolyte and as one of the 20 amino acids used in protein synthesis. Indeed, addition of proline to DARas mutant cells effectively quenched ROS levels and prevented cell death. Treating cells with inhibitors of ROS production yielded similar results. In addition, proline protected wild-type C. trifolii cells against various lethal stresses, including UV light, salt, heat, and hydrogen peroxide. Moreover, proline also protected yeast cells from lethal levels of the ROS-generating herbicide methyl viologen (paraquat), further supporting proline's protective role in response to oxidative stress. The ability of proline to scavenge intracellular ROS and inhibit ROS-mediated apoptosis is an important discovery.

Thus, in one aspect, the invention concerns patentable compositions comprising a carrier and an effective amount of proline or an anti-oxidant proline analog, or a salt thereof.

Another aspect concerns methods of treating or preventing an adverse effect associated with an environmental stress. Such methods comprise administering an effective amount of proline or an anti-oxidant proline analog, preferably in a composition according to the invention, before, during, or after a stressful event.

A related aspect concerns reducing intracellular ROS levels in eukaryotic cells by administration of a an effective amount of proline or an anti-oxidant proline analog, preferably in a composition according to the invention in response to, er in expectation of encountering, elevated intracellular ROS levels as can, for example, occur in response to exposure to an environmental stress.

These and the other methods of the invention can be delivered to any eukaryotic organism, including whole organisms, for example, animals (particularly mammals, including humans) and plants, as well as cells. For example, proline, or an anti-oxidant proline analog, can be administered to cells in culture, including cells (e.g., yeast) being used in fermentation processes, to produce recombinant proteins, monoclonal antibodies, enzymes for industrial applications, etc.

These and other aspects of the invention will be apparent to those skilled in the art upon consideration of the following description of the invention.

BRIEF DCESCRIPTION OF THE FIGURES

This patent application contains at least one figure executed in color. Copies of this patent application with color drawing(s) will be provided upon request and payment of the necessary fee.

Figure 0 shows the generation of different ROS by energy transfer or sequential univalent reduction of ground-state triplet oxygen.

Figure 1 shows the effects of proline on hyphal morphology and intracellular ROS production of both WT and DARas mutant on minimal medium. (A) Cell morphology of WT and the DARas mutant after 6 days of growth on minimal medium with (1.6 mM) or without proline. (B) WT and the DARas mutant were grown at room temperature in minimal medium amended with or without proline. After 6 days of incubation, protoplasts were generated from each strain, and aliquots of protoplast cells were incubated with 50 μM H₂O₂-sensitive fluoropliore 2',7^l-dichlorofluorescindiacetate and then photographed with an epifluorescence microscope. Pictures shown are representative of three independent experiments. Bars represent 20 μm.

Figure 2 shows that proline protects DARas mutant cells against heat stress and yeast cells against paraquat. (A) Conidia of DARas mutants were pretreated at 55°C for 30 min. and then inoculated to minimal medium supplemented with or without proline. Pictures shown are representative of three independent experiments. (B) Yeast cells were inoculated to minimal vitamin medium containing 1 mM MV (paraquat), in the presence or absence of 1.6 mM proline. The colonies were photographed 4 days after inoculation at 30°C. The experiment was repeated in triplicate.

Figure 3 shows that proline inhibits apoptotic responses in DARas mutants. (A) The DARas mutant strain was grown for 6 days on minimal medium with or without proline. Hyphae were treated with Evans blue dye (0.4%) for 24 hr. After extensive washes with PBS, the samples were observed by light microscopy. Pictures shown are representative of three independent experiments. (B) Spores of DARas mutant (Upper) and WT strain (Lower) were grown for 6 days in minimal medium with or without proline and were fixed and stained with DAPI to visualize DNA. (Q DNA fragmentation of DARas mutant cells. Cells were grown as in panel (A), followed by TUNEL assays. (D) Protoplasts produced from the same cultures as in panel (A) were stained for PS with FITC-conjugated annexin V. Only annexin(+)/PI(-) cells underwent apoptotic cell death. One protoplast showing this phenotype was enlarged for observation (Upper Left, upper right corner). The arrow indicates a cell undergoing necrosis, because it also stains for PI. In this figure, the bars represent 20 μm. Figure 4 shows that proline inhibits the apoptotic-like cell death triggered by UV and H 2O2in WT strains. (A) After 24 hr. of incubation on minimal medium with or without proline, the WT hyphae were exposed to UV for 1 min., and a TUNEL analysis was performed. Fluorescence micrographs showed TUNEL (+) and TUNEL (-) hyphae in response to addition of proline. (B) Hyphae of WT strains were collected from medium with or without proline amendment and treated with 1 mM H₂O₂ for 6 hr. PS exposure was assessed by using a FITC- conjugated annexin V binding assay. (Inset) An enlarged apoptotic cell positively staining with annexin V. The bar represents 20 μm.

Figure 5 shows that addition of proline results in rapid and prolonged induction of CAT activity but does not affect SOD activity. (A) CAT activity of C. trifolii WT and DARas mutant strains when grown in minimal medium with or without proline. CAT activity was measured spectrophotometrically by absorbance at 240 nm. (B) SOD activity was measured by the nitroblue tetrazolium reduction. Results indicate the mean and SD from three independent experiments.

Figure 6 shows that proline protects DARas mutant cells against UV and salt stresses. (A) Conidia from wild-type (WT) and DARas mutant strains were plated at 100 conidia per plate on minimal medium amended with or without proline (1.6 mM). After UV irradiation, the number of survivors on each plate was determined after 3 days of incubation. Percent viability represents the percentage of growing colonies remaining in the treated plates as compared with the untreated control plates. Each data point represents the average of four plates. Experiments were repeated three times, and representative data are shown. (B) As in panel (A), conidia were directly inoculated to salt-containing minimal medium at indicated concentrations, with or without proline.

Figure 7 shows that proline inhibits programmed cell death triggered by salt, heat, and hydrogen peroxide (H₂O₂), as assessed by TUNEL assays. Spores of WT strains were inoculated into microscope coverslips overlaid with a thin layer of agar medium with or without proline. After 24 hr. of incubation, the hyphae were treated with 0.5 M NaCl for 2 h (A); 55⁰C for 30 min (B); or 1 mM H 2O2for 6 hr. (Q. After an additional 30 min, DNA damage was assessed by TUNEL assays. The bars represent 20 μm.

As those in the art will appreciate, the following description describes certain preferred embodiments of the invention in detail, and is thus only representative and does not depict the actual scope of the invention. Before describing the present invention in detail, it is understood that the invention is not limited to the particular processes, compositions, formulations, and food items described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention defined by the appended claims.

DETAILED DESCRIPTION

The following detailed description of the embodiments of the present invention, is not intended to limit the scope of the invention, as claimed, but is merely representative of the presently preferred embodiments of the invention. In summary, as described below, this invention is based on the discovery of a novel antioxidation system, namely the amino acid proline acting as a potent intracellular scavenger of ROS. Unlike other amino acids, proline has a cyclized amino nitrogen that has significant influence on the conformation of peptides and polypeptides. Proline is also a major component of structural proteins in animals and plants. As described herein, the previously unappreciated ROS-scavenging property of proline can be used to prevent or reverse the induction of programmed cell death by ROS generated during stress, including biotic and abiotic stresses such as nutritional stress, UV light, cold, heat, high salt, and hydrogen peroxide exposure.

1. Introduction.

Fossil records suggest that bacteria developed the ability to photosynthesize 3,500 million years ago (mya), initiating a very slow accumulation of atmospheric oxygen. Recent geochemical models suggest that atmospheric oxygen did not accumulate to levels conducive for aerobic life until 500-1,000 mya. The oxygenation of Earth's atmosphere resulted in the emergence of aerobic organisms followed by a great diversification of biological species and the eventual evolution of humans.

Although oxygen is thought to have been responsible for the expansion of life on Earth, there are two sides to this molecule: life giving and life taking. Oxygen in the air that animals and plants breathe is a relatively nonreactive chemical. However, when oxygen is exposed to high-energy or electron-transferring chemical reactions, it can be converted to various highly reactive chemical forms (Fig. 0), collectively designated "reactive oxygen species" (ROS). ROS are toxic to biological organisms, as they oxidize lipids, proteins, DNA, and carbohydrates, resulting in the breakdown of normal cellular, membrane, and reproductive functions. Ultimately, toxic levels of ROS can cause a chain reaction of cellular oxidation, resulting in disease and lethality. ROS are unavoidable byproducts of biochemical pathways, such as glycolysis and photosynthesis, central to energy production and storage strategies in aerobic microbes, animals, and plants. As a result, aerobic organisms have evolved enzymatic and non- enzymatic anti-oxidation mechanisms to degrade ROS and avoid oxidative destruction. The growth and reproduction of all aerobic prokaryotes and eukaryotes require a balance between the generation of ROS and the capacity of anti-oxidation systems to eliminate them.

Also when organisms are exposed to abiotic stresses such as temperature extremes, dehydration, salt, UV light, ozone, and heavy metals, ROS are produced. In fact, the generation of ROS is the only event known to be common among such divergent stresses. When an abiotic stress induces an oxidative intracellular environment, organisms produce anti-oxidation systems to decrease the concentration of toxic intracellular ROS.

The ROS story is complicated by the fact that plants and animals also have evolved mechanisms that capitalize on the toxic property of ROS to combat pathogens. For example, when plants are exposed to microbial pathogens, they produce ROS that induce programmed cell death in the plant cells surrounding the infection site to-effectively "wall off' the pathogen and terminate the disease process. ROS may also be transmitted through the phloem to distant plant tissues signaling a pathogen attack. In these examples, ROS act locally as toxin and distantly as signaling molecules. However, it appears that ROS have a number of other biochemical functions, such as biochemical signaling, gene expression, protein inhibition, environmental sensing, and activation of transcription factors.

The role of reactive oxygen species (ROS) in cell communication, control of gene expression, and oxygen sensing is well established. Inappropriate regulation of ROS levels can damage cells, resulting in a diseased state. In Colletotrichum trifolii, a fungal pathogen of alfalfa, the mutationally activated oncogenic fungal Ras (DARas) elevates levels of ROS, causing abnormal fungal growth and development and eventual apoptotic-like cell death, but only when grown under nutrient-limiting conditions. Remarkably, restoration to the wild-type phenotype requires only proline. Thus, this specification describes a previously unrecognized function of proline, namely, its ability to function as a potent antioxidant and inhibitor of programmed cell death, in addition to its well-established role as an osmolyte and as one of the 20 amino acids used in protein synthesis. Indeed, addition of proline to DARas mutant cells effectively quenched ROS levels and prevented cell death. Treating cells with inhibitors of ROS , production yielded similar results. In addition, proline protected wild-type C. trifolii cells against various lethal stresses, including UV light, salt, heat, and hydrogen peroxide. Moreover, proline also protected yeast cells from lethal levels of the ROS-generating herbicide methyl viologen (paraquat), further supporting proline's protective role in response to oxidative stress. The ability of proline to scavenge intracellular ROS and inhibit ROS-mediated apoptosis is an important discovery.

2. Preferred Embodiments.

The small GTP-binding protein Ras regulates cellular signal transduction processes leading to cell growth, differentiation, and survival (1). In mammals, the importance of Ras in regulating growth is underscored by the observation that mutations conferring constitutive Ras activation are found in nearly 30% of all human tumors (2). Moreover, expression of constitutively active Ras in primary cells generally leads to cell-cycle arrest or apoptosis (3). Recently, the role of Ras in filamentous fungi has been studied. Truesdell et al. (4) found that an activating mutation of the unique ras gene (Ct-ras ) from Colletotrichum trifolii, a fungal pathogen of alfalfa, causes— oncogenic phenotypes in nu/nu mice, suggesting that this fungal ras gene has the genetic capability to function as a bona fide oncogene. More interestingly, we found that the dominant activated "oncogenic" Ras (denoted DARas), when expressed in C. trifolii, yielded a^'nutrient- dependent response. Under conditions of nutrient deprivation (minimal medium), the DARas mutant induced aberrant hyphal proliferation, defects in polarized growth, and, significantly, reduced differentiation such as conidiation and appressorium formation (4). Because these mutants showed normal hyphal growth and development in rich medium, it is possible that Ct- Ras regulates a signal transduction pathway that senses and responds to nutrients, similar to what has been observed in Saccharomyces cerevisiae (5). Growth of C. trifolii in minimal medium with various regimes of carbon, nitrogen, heat, and osmoticum failed to complement the DARas mutant (6). Based on the observation that peptone restored the wild-type (WT) phenotype, it was discovered that proline alone, when added to minimal medium at the concentration found in peptone (1.6 mM), was sufficient to fully revert the WT phenotype, including restoration of normal hyphal morphology, polarized growth, and conidiation (Fig. 1) (6).

Proline differs from all other standard amino acids in that it is an α-imino acid. Proline is an osmoprotectaiit in plants, able to balance drought stress (7). In a variety of plants, stresses such as cold, heat, salt, drought, UV, and heavy metals significantly increase endogenous proline concentrations (7,8). One common feature from these stresses is the production of reactive oxygen species (ROS). ROS encompass a variety of partially reduced oxygen metabolites (e.g., superoxide anions, H₂O₂, and hydroxyl radicals) and mediate diverse effects on normal cell functions (9). Of particular note is that mito genie signals induced by activated Ras are mediated by ROS production (9,10). ROS may act as second messengers to induce signaling cascades required for the proliferative response to oncogenic Ras (10). Consistent with this observation, it has been shown that DARas mutant, but not the WT strain, harbors high amounts of intracellular ROS as determined by 2',7'-dichlorodihydrofluorescein diacetate fluorescence when grown in minimal medium (11). In this strain, ROS generation was via a Ras/Rac/cPLA2-dependent pathway (11). Treatment of the DARas mutant with inhibitors of ROS production such as N- acetyl cysteine or diphenylene iodonium decreased ROS levels and concomitantly restored the WT phenorype, similar to what was observed with proline addition (11). These findings thus indicate that ROS production contributes to the aberrant hyphal morphology observed in the DARas mutant grown in minimal medium, which proline can restore to normal growth by reducing intracellular ROS levels.

To study the mechanism of phenotypic restoration of DARas mutant by proline, a DARas C. trifolii mutant was cultured in minimal medium and treated with proline, which significantly inhibited intracellular ROS production. Moreover, high amounts of ROS induced by DARas triggered an apoptotic-like programmed cell death (PCD), as indicated by the appearance of characteristic apoptotic features, including DNA condensation and DNA fragmentation as well as phosphatidylserine (PS) externalization. Importantly, proline prevented this apoptotic response functioning in a cytoprotective manner. In addition, it was found that various stresses, including UV light, salt, heat, and H₂O₂, promote an apoptotic-like PCD when WT C. trifolii was exposed to these treatments and proline inhibited these stress-induced apoptotic responses. Finally, the broad protective role of proline was confirmed by repeating the experiments and obtaining comparable results in the budding yeast S. cerevisiae. Put simply, proline protected yeast cells from lethal levels of ROS generated by paraquat. Taken together, these results establish that proline can function as a potent antioxidant to scavenge intracellular ROS generation and thereby inhibit ROS-mediated apoptotic-like PCD, which may be an important and general function of this amino acid in response to cellular stress, in addition to its well established role as an osmolyte.

3._ Compositions.

Proline, or an anti-oxidant analog of proline, may be delivered in any suitable foπn. Preferred forms are compositions comprising a earner and an effective amount of proline or antioxidant analog of proline, or a salt thereof.

EXAMPLES

The following examples are provided to illustrate certain aspects of the present invention and to aid those of skill in the art in practicing the invention. These examples are in no way to be considered to limit the scope of the invention in any manner. References are indicated by numbers in parentheses, the corresponding citations for which appear at the end of the examples.

These examples demonstrate that the amino acid proline can prevent stress-induced apoptosis using the alfalfa fungal pathogen Colletotrichum trifolii as a representative model system. Previous research had shown that mutationally activated oncogenic fungal Ras (DARas) elevated ROS levels and caused abnormal fungal growth. Below, a C. trifolii mutant that expressed DARas was used, and it was discovered that these fungi reverted to a wild-type phenotype when grown in proline-enriched media. A decrease in intracellular ROS levels in DARas fungi was detected when proline was added to the growth media. Classical markers of apoptosis were monitored in the cells, including DNA condensation, DNA fragmentation, and phosphatidylserine extemalization. All three markers for apoptosis decreased when the mutant fungi were grown with proline. To test proline's ability as a general antioxidant, yeast cells were cultivated in the- presence of paraquat, a herbicide that causes lethal levels of ROS. The paraquot-treated yeast grown in a proline-enriched media survived, whereas controls did not. Thus, proline can be delivered to animals and plants, including animal or plant cells in culture, to combat toxic levels of ROS induced by abiotic or biotic stress. Also, transgenic plants and other eukaryotes can be engineered to have altered levels of intracellular proline (preferably increased levels), particularly in response to, or anticipation of, encountering one or more abiotic or biotic stresses in order to combat the toxic effects of stress-induced ROS.

I. Materials and Methods

1. Strains. The following strains were used in this study: WT C. trifolii race 1 (12); a DARas mutant (a wild-type C. trifolii strain transformed with a dominant active form of Ct-Ras, as described (4)); and wild-type S. cerevisiae strain HAO (MATa).

2. Medium and Growth Conditions. C. trifolii cultures were routinely grown at 25°C on yeast extract-phosphate-soluble starch agar medium or Czapek-Dox minimal medium (0.2% sodium nitrate/0.1% potassium phosphate dibasic/0.05% magnesium sulfate/0.05% potassium chloride/0.001% ferric sulfate/2% agar). When needed, proline was added to the medium at a final concentration of 1.6 mM. S. cerevisiae strain HAO was maintained at 3O⁰C in Minimal Vitamin medium (0.15% Difco Bacto Yeast Nitrogen Base without amino acids/0.52% ammonium sulfate/2% dextrose/2% agar). When needed, methyl viologen (MV; paraquat) or proline was added to the medium at the indicated concentration.

3. Stress Treatments and Viability Assays. Conidia from the appropriate strains were diluted and treated in one of the following ways.

A. For UV viability assays, conidia were plated at 100 per plate on minimal medium amended with or without 1.6 mM proline and allowed to germinate for 3 h before UV irradiation. Plates were incubated for 3 days at room temperature, and the number of survivors on each plate was counted.

B. For salt stress, conidia (10 4per milliliter) were directly plated on minimal medium containing the appropriate concentrations of sodium chloride, in the presence or absence of 1.6 mM proline.

C. For heat stress, conidia (10 6per milliliter) were exposed to heat (55°C) for 30 min and then immediately plated on minimal medium amended with or without 1.6 mM proline.

D. Viability was determined as the percentage of colonies on treated plates compared with untreated controls. For chemical stress in yeast, early logarithmic phase yeast cultures ( A600 = 0.5) were diluted to a density of .4600 = 0.05, and then an aliquot of yeast cells (5 μl) was plated out on MV plates amended with paraquat (1 and 2 mM), proline (1.6 mM), or both. The viable colonies were photographed 3 days after inoculation at 30⁰C. All assays were carried out in triplicate.

4. Detection of Intracellular H₂O₂. Intracellular H₂O₂ levels in C. trifolH were monitored with the oxidant-sensitive probe 2',7'-dichlorofluorescin diacetate (Molecular Probes), as described (11).

5. Evans Blue Staining. Conidia of DARas were inoculated to coverslips overlaid with a thin layer of minimal medium, in the presence or absence of 1.6 niM proline. After 6 days of incubation at room temperature, the cultures were incubated with 0.05% Evans blue for 45 min. at room temperature and washed with PBS. Both pro line-treated and untreated hyphae were observed by light microscopy.

6. DAPI Staining. Nuclei to be observed by fluorescence microscopy were stained with DAPI. After 6 days of growth, the DARas mutant cells were fixed briefly in 70% (vol/vol) ethanol and incubated with 1 μg/ml DAPI in PBS for 15 min. at room temperature, rinsed twice with PBS, and then observed under an epifluorescence microscope (Zeiss Axioskop).

7. TUNEL. TUNEL reactions were performed using the In Situ Cell Death Detection kit (Roche Diagnostics), as described (13). Propidium iodide (PI) staining was used to identify the nuclei.

8. Annexin V Staining. To examine cellular integrity and PS externalization, C. trifolii protoplasts were stained with PI and FITC-conjugated annexin V by using the Annexin V—FITC Apoptosis Detection kit (Oncogene Research Products, Boston). PI is a fluorochrome that cannot cross the membrane of living cells. However, PI can readily penetrate dead cells to stain DNA. Annexin V binding assays were carried out as described (13). Each assay was repeated at least three times.

9. Measurement of Antioxidant Enzymes Activity. CAT activity was determined spectrophotometrically by monitoring disappearance OfH₂O₂ at 240 nm (14). Superoxide dismutase (SOD) activity was measured by the nitroblue tetrazolium reduction, as described (15).

IL Results

1. Proline Inhibits ROS Production by the DARas Mutant on Minimal Medium. Data showing that proline alone, when supplemented to the DARas mutant, is sufficient to restore a WT (wild-type) hyphal phenotype under nutrient-limiting conditions (6), is presented in Fig. IA. It was also found that that the DARas mutant, when grown in minimal medium, produced high amounts of ROS that contribute to the aberrant hyphal morphology because treatment of the DARas mutant with inhibitors of ROS production, such as N- acetyl cysteine or diphenylene iodonium, decreased ROS levels and concomitantly restored the WT phenotype, similar to addition of proline (U ). Moreover, when the DARas mutant was treated with proline analogs (e.g., thiazolidine-2-carboxylic acid, D-proline, 2-azetidinecarboxylic acid, and thiazolidine-4- carboxylic acid), only thiazolidine-4-carboxylic acid mimicked the effect of proline (6). Interestingly, thiazolidine-4-carboxylic acid also is an antioxidant (16). Thus, proline was acting as a ROS scavenger, explaining the ability of proline to revert the activated ras phenotype. This activity was confirmed by monitoring the intracellular ROS levels of the DARas mutant on minimal medium with or without proline, by using 2',7'-dichlorodihydrofluorescein diacetate, a cell-permeable ROS indicator that penetrates live cells but does not fluoresce unless oxidized by ROS (17). As expected, supplementation with proline decreased ROS production by the DARas mutant (Fig. IB). These results show that proline can ameliorate oxidative stress in eukaryotes.

2. Proline Protects DARas Mutant Cells Against Various Abiotic Stresses. In plants, a positive correlation between free proline accumulation and osmotic stress tolerance has been well documented (7). Osmotic stresses, including those caused by drought, salinity, cold, and UV radiation, are tightly linked with ROS generation (18). To test whether praline also protects fungal cells from various abiotic stresses, spores derived from both WT and DARas strains were exposed to heat (Fig. 2 A), UV, or salt (Fig. 6) stress and then grown in minimal medium amended with or without proline. Viability assays indicated the following: (/) DARas strains were more sensitive to these stresses than WT; and (ii) proline protected both WT and the DARas mutants against these stresses, although more significant protection occurred with the mutants. These results indicate involvement of proline in the physiology of stress protection in eukaryotes such as C. trifolii.

3. High Amounts of ROS Induced by DARas Trigger an Ap opto tic-Like PCD, and Proline Inhibits This Apoptotic Response. A useful assay for identifying anti-oxidants is based on the phenotypic restoration of the DARas mutant, which can produce relatively high amounts of ROS may thus induce a PCD-like apoptosis. Treatment with an anti-oxidant, for example, proline, can inhibit/limit this apoptotic response by reducing oxidative stress. Evans blue staining was first used to evaluate the membrane integrity of the DARas mutant with or without proline. Evans blue is a membrane-impermeable stain in normal, healthy cells, but readily penetrates the membranes of dead cells. In minimal medium, Evans blue did not stain germ tubes and young hyphae (Fig. 3 A), but it did stain older hyphae of the DARas mutant (Fig. 3 A). In contrast, growth on minimal medium plus proline prevented Evans blue accumulation in all developmental stages (Fig. 3 A). As a control, no Evans blue staining was detected in the WT strain with or without proline (Fig. 3 A). Thus, the aberrant hyphal growth induced by activated Ras is associated with increased cell death that is inhibited by proline. Use of this and similar systems that produce, or can be induced to produce, toxic intracellular ROS levels, can be used to screen (up to and including high throughput formats) for anti-oxidants, be they administered exogenously or produced by the cells themselves, for example, by way of insertion of a transgene that codes for or otherwise results in the expression of one or more genes related to the desired anti-oxidant (e.g., proline, catalase, SOD, etc.)

Although Evans blue is a reliable vital stain, it does not distinguish between necrotic or apoptotic (programmed) cell death. As already described, apoptosis is a genetically controlled type of PCD characterized by distinct morphological and biochemical changes, including cell shrinkage, chromatin condensation, DNA fragmentation, and membrane externalization of PS on the cell surface (20). These morphological features serve as diagnostic markers for apoptosis. To determine whether ROS induced by DARas triggered apoptotic-like responses in C. trifolu, parameters such as chromatin condensation, DNA fragmentation, and PS externalization can be assayed using any suitable technique. For example, DAPI staining assays can be used to visualize DNA and nuclear morphology. Here, when DAPI staining was performed, DARas cells grown on minimal medium were found to have diffuse nuclear staining, indicating chromatin fragmentation, whereas cells grown on proline-containing minimal medium were like WT cells, displaying compact single nuclei (Fig. 3 B).

DNA fragmentation is another commonly used marker for apoptosis, and is generally detected in situ by the TUNEL assay (21). Strong TUNEL staining was observed in the hyphae of the DARas strain on minimal medium (Fig. 3 C). In contrast, staining was only rarely detected in similar hyphae pretreated with proline (Fig. 3 Q. Thus, the majority of the DARas cells exhibited TUNEL staining under nutrient-limiting conditions, and proline inhibited DNA fragmentation.

Another hallmark of apoptosis is the "flipping", or externalization, of PS from the inner to the outer surface of the plasma membrane. Once exposed, PS can be detected by binding of annexin V to the cell surface (13). As with the TUNEL results, FITC-annexin V binding to fungal protoplasts derived from the DARas mutant were observed, but no staining in proline- treated cells (Fig. 3D) was detected. These observations support proline's function as an active cytoprotectant to suppress an ROS-induced apoptotic-like PCD in the DARas mutant.

4. Proline Inhibits Apoptotic-Like PCD in WT C. trifolii Cells When Exposed to Lethal Abiotic Stresses. PCD has been observed in various organisms when exposed to a variety of abiotic stresses, including UV irradiation, salt, and heat (22 -24). Because proline conferred protection to C. trifolii under each of these stresses, whether these stresses also could induce apoptosis in WT C. trifolii and whether the apoptotic responses could be suppressed by proline were also investigated. By using TUNEL assays, the WT C. trifolii cells, when exposed to lethal levels (determined by Evans blue staining, data not shown) of UV (Fig. AA), salt, or heat (Fig. IA and B), displayed fragmented DNA. No cell death above background occurred under the same conditions in the presence of proline, further demonstrating that proline inhibits apoptosis triggered by UV, salt, or heat.

To further study proline's protective effects on C. trifolii cells against ROS-mediated cell death, WT C. trifolii cells were treated with 1 mM H₂O₂. Again, both TUNEL (Fig. 1C) and annexin V staining assays (Fig. 4 B) indicated AaTH₂O₂ treatment rapidly induced apoptosis in WT cells, and that proline prevented the apoptotic response, indicating that proline ameliorates oxidative stress and prevents PCD.

5. Catalase (CAT), but Not SOD, May Mediate Proline-Dependent Protection in C. trifoUL Antioxidants, including CAT and SOD, protect cells against oxidative stress by maintaining ROS such as H₂O₂ at low levels. The loss of SOD enzyme activity was sufficient to induce apoptosis in cultured motor neurons (25), and CAT prevented apoptosis of the human CEM T cell line in serum-free medium (26), consistent with antioxidant effects on apoptosis. Given these results, the status of the activity of these scavenging enzymes in DARas strains with and without proline was investigated. After growth in minimal medium for 6 days, when compared to the WT strain (which harbored extremely low concentrations of ROS), the DARas cells (which harbor significantly higher concentrations of ROS under these nutrient-limiting conditions) showed a slight increase in CAT activity. Interestingly, treatment of DARas cells with proline caused a nearly 4-fold increase in CAT activity compared with untreated cells, and this high CAT activity was maintained for up to 14 days (Fig. 5 A). SOD activity in the DARas mutant was consistently higher than that of the WT (Fig. 5 B), although the addition of proline did not increase SOD activity (Fig. 5 B). In fact, SOD activity of the DARas mutant was similar to WT levels after 14 days of incubation, independent of proline addition (Fig. 5 B). These data show that proline is related to CAT activity, but not SOD activity, during oxidative stress. 6. Proline Protects Yeast Cells Against Paraquat Killing. To further extend the explore the generality of the anti-oxidant effects of proline, yeast cells were treated with MV (paraquat), a contact herbicide that uncouples electron transport, thereby generating lethal intracellular levels of ROS (27). WT yeast cells treated with 1 mM MV were unable to grow, whereas incubation of MV-treated yeast cells with proline restored normal growth (Fig. 2 B).

III. Discussion

Proline has been discovered to be a potent ROS scavenger associated with prevention of apoptotic-like PCD in cells having elevated levels of intracellular ROS, as compared to cells of the same type cultivated under normal, or non-stressful, conditions. In the context of C. trifolii having a DARas mutation, growth on minimal medium plus proline significantly suppressed intracellular ROS induced by dominant active Ras and inhibited the progression of a ROS- mediated apoptosis. Moreover, proline also inhibited the apoptotic responses triggered by a variety of abiotic stresses. Importantly, the protective role of proline extends to yeast, as well as other eukaryotes. Indeed, proline protected yeast cells against lethal effects of paraquat, a potent ROS generator. Therefore, the ability of proline to scavenge intracellular ROS and thereby inhibit ROS-mediated apoptosis is reasoned to be a general function of this amino acid (and analogs thereof that also exhibit anti-oxidant effects in any suitable model, particularly a model in which toxic levels of ROS can be induced), in addition to its well established role as an osmolyte.

All aerobic organisms generate ROS as metabolic byproducts, mainly as a result of aerobic respiration. ROS can damage DNA, lipids, and proteins, resulting in cytotoxicity. In mammals, ROS have been viewed as second messengers to influence numerous intracellular signaling pathways, including a variety of Ras-mediated cellular effects (28, 29). Of particular note is that ROS act as a downstream effectors of Ras to potentially mediate or initiate an apoptotic process. Indeed, dominant active Ras-transformed NIH 3T3 cells have ben reported to generate larger amounts of superoxide than normal NIH 3T3 cells under basal conditions. In the yeast S. cerevisiae, the oncogenic Ras2 vail 9 mutant reportedly exhibited significantly higher concentrations of ROS, which caused elevated stress sensitivity, increased oxidative damage, and a reduced replicative lifespan (30). These data suggest a linkage between ROS production and Ras signaling. Moreover, it is reported that dominant active Ras promotes apoptosis in several cell lines, including proliferating Drosophila imaginal tissue (31) and fibroblasts (32). Thus, these studies establish Ras as a modulator of apoptosis by regulating intracellular ROS production. Consistent with these observations, the results reported herein establish that in C. trifolii, dominant active Ras expression also generates high concentrations of ROS that trigger a PCD-like apoptosis under nutrient-limiting conditions. As such, such a system can be used as a positive screen to identify anti-oxidants useful in treating ROS-induced cell damage.

Moreover, when WT C. trifolii was treated with lethal levels of abiotic stresses, including UV radiation, salt, heat, and H₂O₂, apoptosis was induced. PCD has been observed in budding yeast after oxidative stress (13). Stress-induced apoptosis also has been noted in Aspergillus nidulans (33) and Candida albicans (34).

In plants, proline constitutes less than five percent (5%) of the total pool of free amino acids under normal conditions. After stress, this level can increase to up to 80% of the amino acid pool (35). The ability of proline to confer stress protection has previously been accounted for by its recognized osmoprotective functions (7). Transgenic plants that cannot produce proline have reduced stress tolerance (36). In addition, other positive roles of proline have been proposed, which include stabilization of proteins (37), regulation of the cytosolic pH (38), and regulation of the NAD/NADH ratio (39). As reported in this specification, a previously unrecognized function of proline has been discovered: its ability to inhibit, prevent, or reverse, at least in part, ROS-mediated apoptosis in eukaryotes. These results establish that proline functions as a potent antioxidant to scavenge intracellular ROS. produced by DARas. The cytoprotective role of proline is specific because all other amino acids or osmolytes were ineffective. Thus, proline is not simply a by-product of stress defense, but instead is a chemically active compound, crucially involved in the physiology of stress protection. As such, it may be delivered throughout the eukaryotic kingdom to treat prevent or reduce the adverse effects of environmental stresses, whether already encountered or expected to be encountered. Preferred examples of eukaryotes to which proline can be administered include animals, such as mammals, plants, and yeast and other eukaryotic cells used in fermentation and cell culture (e.g., CHO and COS cells).

The mechanism of ROS amelioration by proline is reasoned to be mediated by CAT (catalase). Indeed, as shown above, in the DARas mutant of C. trifolii, CAT₅ but not SOD, mediated the proline-dependent prevention of ROS generation and apoptotic initiation, and CAT activity was significantly induced when the DARas mutant was grown on minimal medium supplemented with proline. This correlation between ROS levels and significant increases in CAT activity support proline's role in mediating a CAT-dependent antioxidant pathway that influences the onset of stress-induced apoptosis in eukaryotic cells. In addition, is was found that exogenous proline increases CAT activity in S. cerevisiae when cells were exposed to H₂O₂.

Proline utilization, other than in protein synthesis, occurs primarily inside the mitochondria, where two nuclear-encoded enzymes, proline dehydrogenase (ProDH) and 1- pyrroline-5-carboxylate dehydrogenase, are required to convert proline into glutamate. Both ProDH and l-pyrroline-5-carboxylate have been reported to exhibit the ability to suppress cell growth and to induce apoptosis in a lung carcinoma cell line. Moreover, ProDH is reportedly able to generate ROS. To determine whether or not ProDH is involved in the protective role of proline in ROS-mediated apoptotic responses, the budding yeast S. cerevisiae was used as a model. Results from this model indicated that overexpression of a yeast ProDH gene, putl, exhibited a significantly higher sensitivity to H₂O₂ and paraquat treatments, but accumulation of proline in putl deletion mutant protected yeast cells from these oxidative stresses. Thus, enzymatic removal of proline results in increased sensitivity to oxidative stress. These results indicate that reducing the expression of one or more enzymes involved in proline utilization, particularly in the in the mitochondria (e.g., proline dehydrogenase and l-pyrroline-5- carboxylate dehydrogenase) prior to or during the period when an environmental stress in encountered, will be useful in enhancing stress resistance in various situations, for example, in cell culture and fermentation.

In summary, proline has been shown to possess a potent cell-protective function by ameliorating oxidative stress. Because many biotic (pathogens) and abiotic (e.g., UV and high and low temperatures) stresses involve oxidative stress and PCD, the ability of proline to quench ROS and function as a cytoprotectant has important implications in the treatment and prevention of stress-induced toxicity, up to and including cell death, across the eukaryotic kingdom. Moreover, abnormalities in proline metabolism have been associated with a number of mammalian diseases. For example, ProDH catalyzes the generation of pro line-dependent ROS and promotes apoptosis in human colon cancer cell line (46) and ProDH mutations have been associated with hyperprolinemia in the schizophrenic patients (20). Thus, treatments that compensate for these deficiencies, whether by proline replacement, interference with the expression of one or more proteins involved in proline utilization (e.g., ProDH), and the like, can be used. Put simply, the results presented in these examples demonstrate that modulation of proline levels is an effective means for protecting cells against environmental insults (e.g., abiotic and biotic stresses) and disease.

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All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the spirit and scope of the invention as defined by the appended claims.

All patents, patent applications, and publications mentioned in the specification are indicative of the levels of those of ordinary skill in the art to which the invention pertains. All patents, patent applications, and publications, including those to which priority or another benefit is claimed, are herein incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

The invention illustratively described herein suitably may be practiced in the absence of any element(s) not specifically disclosed herein. Thus, for example, in each instance herein any of the terms "comprising", "consisting essentially of, and "consisting of may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

Claims

I claim:

1. A composition comprising an effective amount of proline, an anti-oxidant proline analog, or a salt thereof, and a carrier.

2. A method of treating an adverse effect associated with an environmental stress, comprising administering an effective amount of proline or an anti-oxidant proline analog to a eukaryotic cell before, during, or after an environmental stress is encountered.

3. A method of reducing an intracellular ROS level in a eukaryotic cell, comprising administering an effective amount of proline or an anti-oxidant proline in response to, or in expectation of encountering, an elevated intracellular ROS levels.

4. A method according to claim 2 or 3 wherein the eukaryotic cell is a plant cell or an animal cell.