Genetical, developmental, and thermal regulation of antioxidant enzymes in Neurospora

FreeRadicalBiology& Medicine,Vol.9, pp. 23-28, 1990 Printedin the USA.All fightsreserved.

0891-5849/90 $3.00+ .00 © 1990PergamonPressplc

Original Contribution GENETICAL,

DEVELOPMENTAL, A N D THERMAL REGULATION OF ANTIOXIDANT E N Z Y M E S IN NEUROSPORA

KENNETH D. MUNKRES Laboratoryof Molecular Biologyand Departmentof Genetics, UniversitYof Wisconsin, Madison, WI 53706 (Received 26 January 1989; Revised 8 January 1990; Rerevised and Accepted 28 March 1990) Abstract--Three characteristics of the biochemical genetics of antioxidant enzyme regulation in Neurospora and enteric bacteria are analogous. This paper reports two additional analogies:responsiveness to change in respiratory rate or thermal stress. A negative regulatory Neurospora mutant is defective in those responses. Although several differences are noted, the common denominator of the two organisms is probably an oxy-regulon, a global unit of genetic function. The degree of homology of bacterial and fungal antioxidant enzyme regulatory mechanisms at the molecular-genetic and signal transduction levels of organization remains to be examined. The hypothesis that the genetic control of antioxidant enzymes is a prerequisite for cellular differentiation of Neurospora is discussed. Keywords--Free radicals, Oxy-regulon, Oxidative stress, Heat-shock

The genetic regulation of antioxidant enzymes by the fungus Neurospora crassa, an eucaryote, exhibits three characteristics of the procaryotic bacterial regulon. (See Discussion). This paper reports two additional analogous characteristics; namely, responsiveness to developmental changes in respiratory rate or thermal stress. A negative regulatory mutant of Neurospora is defective in those responses. The results provide additional support to the hypothesis that the antioxidant enzyme regulatory mechanisms of the fungus and the bacteria are analogous; however, several differences are noted. The common denominator may be an oxy-regulon. The degree of homology of bacterial and fungal antioxidant enzyme regulatory mechanisms at the molecular-genetic and signal transduction levels of organization remains to be examined. The hypothesis that the genetic control of antioxidant enzymes is a prerequisite for cellular differentiation of Neurospora is discussed.

INTRODUCTION Aerobic cells have many antioxidant enzymes that minimize the flux of toxic, reactive-oxygen molecules such as free radicals and peroxides that arise as by-products of normal metabolism or under environmentally imposed oxidative stresses. J Those molecules are implicated as causal factors in many human diseases, normal aging of diverse organisms, and host-pathogen interaction~; therefore, the question of how cells cope or fail to cope with them is important not only for genetics and cell biology but also for medicine. Recently, a unit of genetic function was discovered in enteric bacteria that serves to cope with the stress of reactive-oxygen molecules. (See Discussion). The system is called the oxy-regulon. A regulon is a global unit of genetic function in which a regulatory gene controls the expression of many genes throughout the genome whose products have related functions. The oxy-regulon controls several antioxidant enzymes such as peroxidases and reductases and responds to the stress of endogenous or exogenous reactive-oxygen molecules by synthesizing of those enzymes.

MATERIALS AND METHODS

Strains The mutant Age-l.7 and wild-type A-l-9 are used.2'4'5"14,15,24The mutant had been backcrossed twice to the wild-type parent. The mutant was pure, as judged by the uniformity of its pleiotropic colony phenotype

Address correspondenceto: Kenneth D. Munkres, Laboratoryof Molecular Biology, 1525 Linden Drive, University of Wisconsin, Madison, WI 53706. 23

24

K . D . MUNKRES

among 200 colonies. Master stocks are stored on culture slants at - 20 °. Fresh cultures are grown for each experiment.

Cultures Conidia are produced, collected, and counted as previously described. L~5 The temperature during conidiation was 20-22 °. Conidia are inoculated in 40 mL of Vogel's 6 minimal medium containing 2% sucrose to 106/mL and incubated at 25 ° or 44 ° on a rotary shaker at 250 rpm for 18-24 h, a time corresponding to lateexponential growth phase. Mycelia are harvested on a Btichner funnel and washed with 100 volumes of icecold distilled water.

Enzyme extraction Exocellular SOD is extracted by methods A o r B . 2'7 For extraction of endocellular protein, conidia or mycelia are suspended in 5 mM morpholinopropane sulfonate buffer, pH 7.6, at 10 ° and ground in a carborundum mill. 2'15 Extracts are clarified by centrifugation at 20 Kg for 20 min at 4 °. Supernatants are removed with a syringe to avoid disturbance of the pellet and a floating lipid layer.

Protein and enzyme assays These methods were described previously. 2'15 Malate dehydrogenase is assayed with oxaloacetate and NADH. 8 All assays are at 36 °. Units of activity are noted in Table 1. Enzymes' abbreviations, trivial and systematic names, and commission numbers are in Ap-

pendix.

RESULTS

Table 1 summarizes activities of 11 enzymes from wild-type conidia and mycelia grown at 20-25 °. The enzymes are grouped according to their more or less known subcellular location. 7,9,13,15(The present extraction procedure disrupts mitochondria. Mitochondrial enzymes are in the 20 Kg supernatant. Subcellular location of all of the enzymes is not established with equal certainty.) The observed large increases of the well-known mitochondrial enzymes MDH and COX are consistent with knowledge that the developmental process is accompanied by mitochondrial biogenesis and a large increase of cellular respiratory rate. 10-12 Constitutive conidial COX 2 and MDH* are not controlled by the age genes. Wild-type mitochondrial CPX and SOD(CNR) increase 170- and 330-fold, respectively, during development. Four of the other seven antioxidant enzymes also increase significantly by 2- to 7-fold. The small increase of the GPX isozymes is probably not significant. Previous experiments indicate that selenite must be added to the culture medium to obtain high constitutive expression of selenium-dependent GPX isozymes. 2 Table 2 compares the specific activities of six antioxidant enzymes from conidia and mycelia of wild type and the regulatory mutant. The results confirm previous observations that, in general, those mutants' conidia are deficient in those enzymes. 2'1s Four of the five enzymes increase by about the same degree in mutant and wild; however, they are also relatively deficient in the mutant's mycelia. *Munkres, K. D. unpublished observations.

Table 1. Developmental Regulation of Enzymes in Wild-type Development stage s

Neurospora

Specific activity b" Subcellular locatiow M

conidia(c) mycelia(m) ratio, m / c

C

E.C.

MDH

COX

CPX

SOD(CNR)

SOD(CNS)

AFR e

CAT

MPX'

GPX

0.29 7.5

2.5 100

12 2,040

2.5 825

3.3 0

4.0 8.7

3.6 8.3

0.056 0.27

108 142

330

- ~

2.2

2.3

4.8

26

40

170

1.3

SOD 185 1,330 7.2

GPX 190 296 1.6

°Conidiation was at 20-25 °. Mycelia were from 18 h aerated cultures at 25°. bSpecific activities are units per rain per mg endocellular protein, except E.C. enzymes which are per mg exocellular protein. Units are listed below. E.C. SOD was extracted with KBr. cM: mitochondria. C: cytosol. E.C.: exocellular. The enzymes' abbreviations, trivial and systematic names, and commission numbers are in Appendix. Enzyme units: MDH, #moles NADH; COX and CPX, nmoles cytochrome c; SOD, 150; AFR, #moles NADH; CAT, gmoles H202; MPX, AA2s0,mand; GPX, nmoles GSH. ~"I'he experiment was performed once. The reproducibility of the assays of a sample is sufficmnt to insure that a ratio(m/c) of at least 2 is statistically significant ( p = 0.1), as indicated by one-side t tests. eEndocellular location uncertain.

Antioxidant enzyme regulation

25

Table 2. Developmental Control of Six Antioxidant Enzymes in Neurospora Wild-type and a Regulatory Mutant Developmental stage*

Specific activityb Enzyme SOD CNS

conidia(c) mycelia (m)

Strain: c

2.6 0

+ 3.3 0

ratio: m/c

-oo

-o0

E.C. SOD

CPX

CAT

AFR

CNR 0.1 15 150

+ 2.5 185 330

11.5 110 9.6

+ 185 1,330 7.2

2.8 <2.2 <1

+ 12 2,040

1.8 5.7

+ 3.6 8.3

1.2 2.2

+ 4.0 8.7

170

3.2

2.3

1.8

2.2

aCells were grown at 20-25° as described in Methods. bSpecificactivitiesare defined in Table 1. Enzymeabbreviationsare defined in Appendix. Statistical significanceof the magnitude of the ratio, m/c, is defined in Table 1. c.: mutantAge-l.7. +: wild-typeA-l-9. Cyanide-sensitive superoxide dismutase is not detected in either wild-type or mutant mycelia, but is detected in both strains' conidia (Tables 1,2); however, on other occasions the enzyme was observed in wildtype mycelia.13'~4 The cause of that discrepancy is unknown. Thus, the developmental control of that enzyme remains relatively uncertain. The mutant's conidial deficiency of that enzyme is, however, a reproducible observation (Table 2). 2 Six antioxidant enzymes of wild-type mycelia grown at 44 and 25 ° were assayed. Only the exocellular superoxide dismutase is correlated consistently with temperature. The wild-type enzyme is reproducibly 190-fold greater at the higher temperature; but the mutant's enzyme is elevated only 3.5-fold (Table 3). Obviously, the relative incapacity of the mutant to respond to thermal stress accentuates its relative deficiency of that enzyme at the higher temperature.

DISCUSSION

Antioxidant enzyme regulatory mechanisms of Neurospora and bacteria Cells of diverse species, ~ including Neurospora, 3 respond to exogenous oxidative stress by synthesizing greater amounts of antioxidant enzymes:a phenomenon that suggests the existence of genetic regulatory mechanisms. The first experimental indication of such a mechanism was noted in Escherichia coli. 16 Subsequently, a regulatory gene oxy-R was discovered in E. coli and the related enteric bacteria Salmonella typhimurium.17 Positive mutation of oxy-R confers resistance to H202 and elevated constitutive activities of five antioxidant enzymes; whereas negative mutation of that gene confers hypersensitivity to H202 and incapacity to synthesize those enzymes either constitutively or in response to exogenous oxidative stress.

Although it is not known whether the positive and negative regulatory gene mutations age + and Age- of Neurospora occur at the same gene(s) 4'24 they are functionally analogous to the oxy-R mutations. Age- and age +, respectively, confer inferior and superior constitutive activities of 12 conidial antioxidant enzymes (Table 2). TM Moreover, Age- and age +, respectively, confer inferior and superior resistance to growing mycelia against five types of exogenous oxidative stresses, as may be expected from their quantitative antioxidant enzyme phenotypes.* Most of the structural genes encoding antioxidant enzymes are not closely linked to the oxy-R locus.17'ts The same situation exists in Neurospora.2 The situation suggests the existence of a trans-acting regulatory protein, but such a protein has not been reported in either the bacteria or the fungus. E. coli and yeast cells adjust superoxide dismutase synthesis in response to change of their respiratory rate. I An increase of respiratory rate leads to an increase of the by-products superoxide radicals. Wild-type Neurospora also exhibits an increase of two SOD isozymes and three other antioxidant enzymes in the developmental transition from dormant conidia to respiring mycelia (Table 1). The observed large increases of the mitochondrial respiratory enzymes malate dehydrogenase and cytochrome oxidase are consistent with the fact that the developmental process is accompanied by mitochondrial biogenesis and a large increase in cellular respiratory rate. ~°-12 (Cytochrome oxidase and malate dehydrogenase are not controlled by the age genes.) 2. The magnitudes of increase of two mitochondrial antioxidant enzymes are much larger than those of cytosolic or exocellular antioxidant enzymes (Table 1). That differential probably reflects that mitochondria *Munkres, K. D. unpublished observations.

26

K . D . MUNKRES Table 3. T h e r m a l and Constitutive R e g u l a t i o n of Exocellular Superoxide D i s m u t a s e is Defective in Mycelia of a R e g u l a t o r y M u t a n t Strain"

E.C. S O D activity b

25 mutant(m) wild type(w) ratio: m / w

C u l t u r e t e m p . (°C) 44

1.02 - 0.08 x 102 1.37 - 0.04 x 103

3.6 - 0.4 × 102 2.6 - 0.1 x 105

0.07

0.001

ratio: 44/25 3.5 190

~Mutant Age-l.7. Wild type A-I-9. Mycelia were grown and extracted as described in Methods. (Method B) bUnits per mg exocellular protein. Average and variance of two independent experiments.

are the main subcellular site of oxidative metabolism and its by-products, free radicals and H202. The two mitochondrial enzymes, superoxide dismutase and cytochrome c peroxidase, catalyze the decomposition of superoxide and H202 in a concerted manner. (Catalase does not occur in the mitochondria.) 15 The existence of mitochondria in eucaryotes probably creates special problems in antioxidant enzyme regulation, particularly during mitochondrial biogenesis when preferential synthesis of the mitochondrial enzymes may be expected (Tables 1,2). In other states of cellular differentiation, such as conidia, the regulatory genes appear to control the synthesis of 12 antioxidant enzymes in a coordinate manner] Such coordination may be required to ensure optimal "collaborative" function of the enzymes. 2 Since enteric bacteria neither undergo cellular differentiation nor have mitochondria, their regulatory mechanism may not be exactly analogous to that of the fungus. An Age- mutant exhibits inferior activities of the antioxidant enzymes at either developmental stage; however, with the exception of cytochrome peroxidase, it also increases those enzymes' activities during development (Table 2). Apparently, synthesis of the enzymes is repressed in both cell types, but the mechanism of response to the developmental process is not inactive. That observation lends additional support to the postulated regulatory role of the genes. Three groups of investigators propose that thermal stress is a form of oxidative stress in enteric bacteria. 17 J9.2~ Oxidative stress induces the synthesis of a large number of proteins that are also synthesized in response to thermal stress, that is, "heat-shock" proteins. The oxy-R gene also regulates the synthesis of some heatshock proteins.17-19 Van Bogelen et al.19 propose that the bacterial oxy- and heat-shock regulons have overlapping specificities of response to regulatory signal(s) in common to the two forms of stress. Relationships between the genetic control of responses to oxidative and thermal stresses also occur in Neurospora. The age genes regulate the constitutive

activity of an exocellular superoxide dismutase isozyme 2 (Table 3). That enzyme is elevated 190-fold in wild-type cells subjected to thermal stress, but the enzyme of an Age- mutant is essentially unresponsive (Table 3). Similarly, synthesis of a nonspecific peroxidase (E.C. 1.11.1.7), regulated by the age genes, 2 is induced in wild-type cells under either H202 or thermal stress.2° Hence, two antioxidant enzymes regulated by the age genes are also heat-shock proteins. Although several analogies between bacterial and fungal antioxidant enzyme regulation are indicated, several differences are also indicated. First, the bacterial Mn-superoxide dismutase is a heat-shock protein, 2~ but the fungal enzyme is not. 2°* Second, contrary to a previous r e p o r t , 17 the bacterial Mn-superoxide dismutase is not regulated by the oxy-R gene, 22 but the fungal enzyme is regulated by the age genes (Table 2). 2'15 Third, the bacteria probably have two oxy-regulons, 23 but thus far only one fungal regulon is known. Fourth, four antioxidant enzymes are controlled by the oxy-R gene, 17A8,22 but 12 are controlled by the age genes (Table 2). 2,15 The degree of homology of procaryotic and eucaryotic antioxidant enzyme regulatory mechanisms at the molecular-genetic and signal transduction levels of organization remains to be examined. The scope of this discussion does not permit consideration of the uncertain nature of the signal transduction mechanism. The biochemical genetic nature of the mechanism by which Neurospora and yeast cells sense free radical / oxidant stress in the regulation of antioxidant enzyme synthesis will be considered in subsequent communications.

Genetic control of antioxidant enzymes: A prerequisite for cellular differentiation? Neurospora is a multicellular organism. Four cell types occur in the life cycle: mitotically active hyphae *Munkres, K. D. unpublished observations.

Antioxidant enzyme regulation

(mycelia); differentiated aerial hyphae, conidiophores and; postmitotic cells, conidia, and ascospores. The genetic regulation of antioxidant enzymes is a determinant of conidial lifespan 2'4'15and probably of cellular differentiation also. Age- mutants are conditionally defective in asexual cellular differentiation. 4'5'24On colonializing medium, Age- colonies fail to form conidiophores and are, therefore, conidiation defective. If, however, they are fed an antioxidant vitamin E, they develop normally. 5. Moreover, an Age- mutant is defective in antioxidant enzyme regulation in the reversible conidial-mycelial differentiation process (Table 2). Those observations indicate that uncontrolled free-radical/oxidant reactions may have a deleterious effect on asexual cellular differentiation. Age- ascospores from the type cross Age- x Ageare usually inviable. 2. When those mutations are at different linked genes, usually only the wild-type recombinant ascospores are viable. Those observations may indicate that uncontrolled free-radical/oxidant reactions also impair sexual cellular differentiation. A subsequent communication will report additional experimental tests of the deleterious role of free radicals in cellular differentiation of Neurospora.

10. 11. 12. 13. 14.

15.

16. 17.

18.

19. Acknowledgments--This research was supported by the University of Wisconsin's College of Agriculture and Life Sciences, the Graduate School, the Laboratory of Molecular Biology, and by Graduate school-administered N1H biomedical research grants. Contribution no. 3078 from the Department of Genetics.

20. 21.

REFERENCES 1. Halliwell, B.; Gutteridge, J. M. C. Free radicals in biology and medicine. Oxford: Clarendon Press; 1985. 2. Munkres, K. D. Genetic co-regulation of longevity and antioxidant enzymes in Neurospora crassa. Free Radical Bio. Med., in press, 1990. 3. K. D. Munkres, Biochemical genetics of aging of Neurospora crassa and Podospora anserina: a review. In: Sohal, R. S., ed. Age Pigments. Amsterdam: Elsevier/North Holland Biomedical Press; 1981:83-100. 4. K. D. Munkres. The role of genes, antioxidants, and antioxienzymes in aging ofNeurospora: A review. In: Oberley, L. W., ed. Superoxide dismutases, Vol. III, Pathological states. Boca Raton, FL: CRC Press, 1985: 237-248. 5. Munkres, K. D.; Furtek, C. A. Selection of conidial longevity mutants of Neurospora crassa. Mech. Age. Dev. 25:47-62; 1984. 6. R. H. Davis; F. J. deSerres. Genetic and microbiological techniques for Neurospora crassa. In: Tabor, H.; Tabor, C. W., eds. Meth. Enzymol., Vol. 17A. New York: Academic Press; 1970:79-143. 7. K. D. Munkres. Purification of exocellular superoxide dismutases. In: Packer, L., ed. Meth. Enzymol., Vol. 186. New York: Academic Press; 1990:249-260. 8. Munkres, K. D.; Richards, E M. The purification and properties of Neurospora malate dehydrogenase. Arch. B iochem. Biophys. 109:466-479; 1965. 9. Beneveniste, K.; Munkres, K. D. Cytoplasmic and mitochon*Munkres, K. D. unpublished observations.

22.

23. 24.

27

drial malate dehydrogenases of Neurospora: regulatory and enzymic properties. Biochem. Biophys. Acta 220:161-177; 1970. Colvin, H. J.; Saner, B, L.; Munkres, K. D. Respiration of wild type and extrachromosomal mutants ofNeurospora crassa. J. Bacteriol. 116:1314-1321; 1973. Sehmit, J. C.; Brody, S. Biochemical genetics of Neurospora crassa conidial germination. Bact. Rev. 40:1-41; 1976. Stade, S.; Bramble, R. Mitochondrial biogenesis during fungal spore germination: respiration and cytochrome c oxidase in Neurospora crassa. J. Bacteriol. 147:757-767; 1981. Munkres, K. D.; Minssen, M. Ageing of Neurospora crassa. I. Evidence for the free radical theory of ageing from studies of a natural-death mutant. Mech. Age. Dev. 5:79-98; 1976. Munkres, K. D.; Rana, R. S. Genetic control of cellular longevity in Neurospora crassa: a relationship between cyclic nucleotides, antioxidants, and antioxigenic enzymes. Age 7:3035; 1984. Munkres, K. D.; Rana, R. S.; Goldstein, E. Genetically determined conidial longevity is positively correlated with superoxide dismutase, catalase, cytochrome c peroxidase, glutathione peroxidase, and ascorbate free-radical reductase activities in Neurospora crassa. Mech. Age. Dev. 24:83-100; 1984 Hassan, H. M.; Fridovich, I. I. Superoxide, hydrogen peroxide, and oxygen tolerance of oxygen-sensitive mutants of Escherichia coli. Rev. Infect. Dis. 1:357-369; 1979. Christman, M. E; Morgan, R. W.; Jacobsen, E S.; Ames, B. M. Positive control of a regulon for defenses against oxidative stress and some heat-shock proteins in Salmonella typhimurium. Cell 41:753-762; 1985. Morgan, R. W.; Christman, M. E; Jacobsen, E S.; Storz, G.; Ames, B. M. Hydrogen peroxide-inducible proteins in Salmonella typhimurium overlap with heat shock and other stress proteins. Proc. Natl. Acad. Sci. USA 83:8059-8063; 1986. Van Bogelen, R. A. ; Kelley, P. M.; Neidhart, E C. Differential induction of heat shock, SOS, and oxidative stress regulons and accumulation of nucleotides in Escherichia coli. J. Bacteriol. 169:26-32; 1987. Kapoor, M.; Lewis, J. Heat shock induces peroxidase activity in Neurospora crassa and confers tolerance toward oxidative stress. Biochem. Biophys. Res. Commun. 147:904-910; 1987. Privale, C. T.; Fridovich, I. Induction of superoxide dismutase in Escherichia coli by heat shock. Proc. Natl. Acad. Sci. USA 84:2723-2726; 1987. Bowens, S.; Hassan, H. M. Induction of the manganese-containing superoxide dismutase in Escherichia coli is independent of the oxidative stress (oxyR-controlled) regulon. J. Biol. Chem. 263:14808-14811; 1988. Greenberg, J. T.; Demple, B. A global response induced in Escherichia coli by redox-cycling agents overlaps with that induced by peroxide stress. J. Bacteriol. 171:3933-3939; 1989 Munkres, K. D.; Furtek, C. A. Linkage of conidial longevity determinant genes in Neurospora crassa. Mech. Age. Dev. 25:63-77; 1984.

APPENDIX

Enzyme abbreviations, trivial and systematic names, and commission numbers. CPX, cytochrome c peroxidase, ferrocytochrome c:hydrogen-peroxide oxidoreductase, 1.1 1.1 5 GPX, glutathione peroxidase, glutathione:hydrogenperoxide oxidoreductase, 1.1 1.1.9 MPX, chloride peroxidase (myeloperoxidase-like), chloride:hydrogen-peroxide oxidoreductase, 1.11.1.10 AFR, ascorbate free-radical reductase, NADH: monodehydroascorbate oxidoreductase, 1.6.5.4

28

K . D . MUNKRES

CAT, catalase, hydrogen-peroxide:hydrogen-peroxide oxidoreductase, 1.11.1.6 COX, cytochrome c oxidase, ferrocytochrome c:oxygen oxidoreductase, 1.9.3.1 SOD, CNR and CNS, cyanide-resistant and sensitive

superoxide dismutases, superoxide: oxidoreductase, 1.15.1.1. E.C. SOD, exocellular SOD MDH, malate dehydrogenase, NADH:malate oxidoreductase, 1.1.1.37.