[13] Measurement of nitric oxide-mediated effects on zinc homeostasis and zinc finger transcription factors

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[13] M e a s u r e m e n t o f N i t r i c O x i d e - M e d i a t e d E f f e c t s o n Zinc Homeostasis and Zinc Finger Transcription Factors

By K.-D. KR~NCKE and V. KOLB-BACHOFEN Introduction Zinc is an essential trace element in eukaryotes. It does not exhibit biologically relevant redox activity, but because of its physical and chemical properties, including its general stable association with proteins and its coordination flexibility, Zn 2+ is highly adaptable to meeting the needs of proteins and enzymes to carry out diverse biological functions. Zn 2+is an integral component of many enzymes, is essential for their catalytical function and structural stability, and is the only metal encountered in each enzyme class. The cytoplasm contains the major number of zinc metalloproteins, but in cell membranes, nuclei and other organelles such proteins serve nonenzymatic functions also. Zn 2+can stabilize functional protein domains named zinc fingers. These have in common tetrahedral ZnZ+-binding sites with four ligands from the side chains of cysteine and/or histidine. The specific local protein conformations of such zinc fingers serve for specific binding of DNA, RNA, or proteins. More than 10 classes of such ZnZ+-based domains have been characterized, 1,2 including the LIM domain3 and the RING-finger motif.4 Transcription factors binding to DNA contain zinc fingers, helix-turn-helix or helix-loophelix motifs, or leucine zippers. However, the zinc finger is by far the most prevalent DNA-binding motif. Destruction of zinc finger domains by oxidation or via chelation of Zn 2+changes the local protein conformation, thereby abrogating its binding specificity for DNA, RNA, or protein. Nitric oxide (NO) has been found to play an important role as a signal molecule as well as a cytotoxic or a regulatory effector molecule. Major relevant targets of NO are transition metals and thiols at catalytical, allosteric, or structural sites of proteins. NO mediates Zn 2+ release from the Zn 2+complexing protein metallothionein and inhibits the DNA-binding activity of the zinc finger-type yeast transcription activator LAC9. 5 Moreover, in 1 j. W. R. Schwabe and A. Klug, Nature Strucr Biol. 1, 345 (1994). 2 j. M. Berg and Y. Shi, Science 271, 1082 (1996). 3 I. S~inchez-Garcfa and T. H. Rabbitts, Trends Genet. 10, 315 (1994). 4 A. J. Saurin, K. L. B. Borden, M. N. Boddy, and P. S. Freemont, Trends Biochem. Sci. 21, 208 (1996). 5 K. D. Kr6ncke, K. Fehsel, T. Schmidt, F. T. Zenke, F. Dasting, J. R. Wesener, H. Bettermann, K. D. Brennig, and V. Kolb-Bachofen, Biochem. Biophys. Res. Commun. 200, 1105 (1994).

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live cells, exogenous N O mediates intracytoplasmic and intranuclear Zn 2+ release in several cell types. 6 We have described methods to investigate interactions of N O with zinc finger proteins in vitro. 7 In this article we describe methods to localize and quantify intracellular free Zn 2+ in living cells after exposure to N O and to study the effects of N O on zinc finger transcription factors in cells. Application of NO in Cell C u l t u r e Working with N O in gaseous form requires special technical equipment. In addition, to administer exact and reproducible N O gas concentrations to cells is difficult to achieve. Fortunately, these problems can be overcome by using chemical N O donors, compounds that chemically generate N O plus an inert compound on dissolving them in aqueous solution. Nitroprusside (sodium pentacyanonitrosylferrate) and SIN-1 are N O donors frequently used. However, both are not r e c o m m e n d e d for use in cell culture. Nitroprusside does not spontaneously generate NO, and SIN-1 generates equal amounts of N O and O{ during decomposition. 8 N O donors r e c o m m e n d e d for studying N O - m e d i a t e d effects on cells are nitrosothiols and 1-substituted diazen-l-ium-l,2-diolates (also known as p o l y a m i n e / N O adducts or NONOates). Nitrosothiols spontaneously decompose to generate N O and the corresponding disulfide. One disadvantage of this N O - d o n o r class is that their decomposition kinetics depends on copper and iron ions as well as on the thiol concentration of the buffer or culture medium. Thus, the exact N O generation values, especially in the presence of cells, cannot be predicted, and may vary between experiments. The big advantage of nitrosothiols is that high yields can be prepared by the reaction of the desired thiol with nitrite at acidic pH, which is a fast, cheap, and easy way to obtain N O donors. The nitrosothiols most often used are S-nitrosocysteine ( S N O C or CysNO), S-nitrosoglutathione ( S N O G or GSNO), and S-nitroso-N-acetylpenicillamine (SNAP). S N O C decays with a half-life in the range of several minutes, while SNAP and S N O G exhibit a half-life of several hours. Synthesis of a 100 m M S N O C stock solution: 9 1. Dissolve 7.04 mg cysteine hydrochloride in 192/xl of doubly distilled H 2 0 on ice. 6 D. Berendji, V. Kolb-Bachofen, K. L. Meyer, O. Grapenthin, H. Weber. V. Wahn, and K. D. Kr6ncke, FEBS Lett. 405, 37 (1997). 7 K. D. Kr6ncke and V. Kolb-Bachofen, Methods Enzymol. 269, 279 (1996). K. D. KrOncke, K. Fehsel, and V. Kolb-Bachofen, BioL Chem. 376, 327 (1995). 9 A. Mirna and K. H. Hofmann, Fleischwirtschafi 49, 1361 (1969).

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2. A d d 2.76 mg NaNO2 dissolved in 192 /xl of ice-cold doubly distilled H20. 3. I m m e d i a t e l y add 8/xl of 1 M HC1 to achieve p H 2 (solution turns deep red). 4. Neutralize with 7 - 8 / z l of 1 M N a O H after 1-2 min. Synthesis of a 100 m M S N O G stock solution: 1° 1. Dissolve 12.28 mg glutathione ( G S H ) in 160/xl of doubly distilled H 2 0 on ice. 2. A d d 2.76 mg NaNO2 dissolved in 160 /xl of ice-cold doubly distilled H20. 3. I m m e d i a t e l y add 40/zl of 1 M HCI to achieve p H 2 (solution turns deep red). 4. Neutralize with about 40/xl of 1 M N a O H . Synthesis of SNAP: 11 1. Dissolve 25 mg of N-acetyl-DL-penicillamine in 300/xl of methanol plus 60/xl of 1 M N a O H on ice. 2. A d d 72.5 mg of NaNO2 dissolved in 100/xl of ice-cold doubly distilled H20. 3. Acidify carefully with about 90 /xl of concentrated (37%) HC1 to achieve p H 1 (a green solid precipitates). 4. Vortex and leave on ice for about 10 rain. 5. Centrifuge and wash the precipitate three times with 200/zl of icecold doubly distilled H20. 6. D r y in vacuum (e.g., over P2010). For applying S N A P to cell cultures, prepare a 50 m M stock solution on ice (1.10 rag/92/xl buffer plus 4/xl of 1 M N a O H plus 4/zl of 1 M HC1). After its synthesis, S N O C should be added to the cells as fast as possible, as it immediately begins to decompose due to its relatively short half-life. In solution, S N O G and SNAP, respectively, are stable for several hours when maintained in the absence of Fe 2+ and Cu 2+ and in the dark. However, to achieve reproducible results, we r e c o m m e n d immediate use here, too. The N O - d o n o r family of the N O N O a t e s are ideal N O donors. U n d e r physiological conditions they spontaneously release N O with predictable kinetics. 12 The most popular N O N O a t e s are D E A / N O (rl/2 at 37 ° = 2 10Commercially available GSH may contain various amounts of oxidized (dimeric) GSSG; thus, caution should be taken to use pure reduced GSH. 11Modified from L. Field, R. V. Dilts, R. Ravichandran, P. G. Lenhert, and G. E. Carnahan, J. Chem. Soc., Chem. Commun., 249 (1978). 12L. K. Keefer, R. W. Nims, K. M. Davies, and D. A. Wink, Methods Enzymol. 268, 281 (1996).

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min), S P E / N O (~'~/2 = 40 min), and D E T A / N O (TI/2 in our hands is about 8 hr). Synthesis of N O N O a t e s should be performed by experienced chemists only as they have to be synthesized under anaerobic conditions at a pressure of 5 atm via reaction of N O gas with the corresponding amine. 13 Various N O N O a t e s are commercially available, but unfortunately they are quite expensive. Therefore, most studies on cells using N O donors are performed using nitrosothiols, and N O N O a t e s are often used to reproduce and validate results obtained with nitrosothiols. To ensure that effects found by using a special N O donor are indeed mediated by NO, a negative control should consist of the respective denitrosylated N O donor, prepared by incubation for several half-lives at 37 °. This is essential when using N O N O a t e s , because polyamines exhibit various biological activities. N O at high concentrations will be toxic for cells. However, different cell types differ in their sensitivity toward NO. TMThus, it should be tested with increasing concentrations where induction of apoptosis or necrosis becomes a prominent effect. We usually incubate the cells with 0.1-5 m M of the different N O donors. After 6, 12, and 24 hr, lysis and/or necrosis is determined by trypan blue exclusion, and staining with a D N A stain (e.g., Hoechst 33342 or 33258) reveals apoptosis.

Localization of Zn 2+ Release in Cells after E x o g e n o u s NO Application Methods to detect zinc in biological systems include atomic absorption spectrometry, atomic or X-ray fluorescence, anodic stripping voltametry, ~5 or the use of radioactive 65Zn2+. Two fluorophores have been developed that both specifically bind Zn 2+. T S Q [N-(6-methoxy-8-quinolyl)-p-toluenesulfonamide; Molecular Probes, Eugene, O R ) and Zinquin [ethyl(2-methyl8-p-toluenesulfonamido-6-quinolyloxy)acetate; Luminis Pty Ltd, Adelaide, South Australia, or Alexis, San Diego, CA] have the same chemical backbone. However, Zinquin contains an additional methyl group for better complexation of Zn 2+ and an ester functional group that is hydrolyzed by cellular esterases, thus trapping the charged fluorophore within the cells. The corresponding Zinqnin acid forms both 1 : 1 and 1 : 2 complexes with Zn 2+ with dissociation constants of 4 x 10 -7 and 9 × 10 8 M, respectively. 16 The excitation and emission fluorescence spectra of the Zinquin acid/Zn 2+ ~3j. A. Hrabie, J. R. Klose, D. A. Wink, and L. K. Keefer, J. Org. Chem. 58, 1472 (1993). 14K. D. Kr6ncke, K. Fehsel, and V. Kolb-Bachofen, N O Biol. Chem. 1, 107 (1997). 15K. H. Falchuk, K. L. Hilt, and B. L. Vallee, Methods Enzymol. 158, 422 (1988). 16I. B. Mahadevan, M. C. Kimber, S. F. Lincoln, E. R. T. Tiekink, A. D. Ward, W. H. Betts, I. J. Forbes, and P. D. Zalewski, Ausr J. Chem. 49, 561 (1996).

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complex in buffer show single peaks with broad maxima in the range of 364 and 485 nm, respectively. 17'18 Zinquin acid does not form a fluorescent complex with other divalent cations with the exception of Cd 2+, and, with the exception of Cu e+, none of these metals interferes with the fluorescence of the Zinquin acid/Zn 2+ complex, even at a 100-fold molar excess. 16'17 Although neither Cd 2+ nor Cu 2+ are major constituents of cells, it may be that local organelle environment can interfere in rare conditions, but this has not been noted so far. When incubating cells with NO donors and studying intraceUular Zn 2+release, it proved useful to culture in the presence of 2.5% fetal calf serum (FCS) instead of the usual higher concentrations. At low FCS concentrations we found maximal intracellular Zn 2+ staining. This may be due to a NO-scavenging effect of FCS, to an esterase activity in culture supernatants, or to complex formation of the fluorophore with serum constituents. To visualize labile intracellular Zn 2+, cells were labeled with the Zn2+-specific fluorophores Zinquin or TSQ. Neither Zinquin nor TSQ show any fluorescence after incubation with NO gas, nitrosothiols, or NONOates in the absence of Zn 2+, nor do the NO donors or NO gas interfere with Zn > dependent fluorescence. At the end of the cell culture time, Zinquin [stock solution: 5 m M in ethanol or dimethyl sulfoxide (DMSO)] is added directly to the cells (final concentration: 25/zM), and these are incubated for 30 min at 37 °. When using TSQ (stock solution: 3 mM in DMSO), this is also added directly to the cell cultures (final concentration: 30 ~M), and these are then incubated for about 15 min at 4 °. Subsequently, live cells can be investigated directly under a fluorescence microscope (excitation in the U V range at about 300-400 nm, emission at about 450-550 nm) (see Fig. 1). Depending on the emission filter, fluorescence is blue to green. Treating live L929 fibroblasts or primary endothelial cells with 5-10 m M S-nitrosocysteine (SNOC) for about 1 hr or with 1-2 m M D E T A / N O for 24 hr, Zn2+-specific fluorescence can be observed in the cytoplasm and also in the nuclei. 6 Treatment of cells with up to 5 m M H202 does not induce any intracellular Zn 2+ release within 1 hr, as an additional proof that this effect is NO-specific. As a positive control, cells can be loaded with Zn 2+ via addition of 100/zM of a Zn(II)-histidine complex (dissolve 25 m M ZnCI2 and 50 m M L-histidine hydrochloride in 1 ml of H 2 0 and neutralize with 1 M NaOH). As negative controls, denitrosylated NO donors (SNOC_No, DETA/NO_No) should be used. In addition, the zne+-specific 17 p. D. Zalewski, S. H. Millard, I. J. Forbes, O. Kapaniris, A. Slavtinek, W. H. Betts, A. D. Ward, S. F. Lincoln, and I. Mahadevan, J. Histochem. Cytochem. 42, 877 (1994). 18 Product information sheet.

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Cell culture m mediumwith< 2.5 % FCS

exposureto NO via NO-donor for desiredtimespan

Addition ofZnZ+-specificfluorophore

I

TSQ

Zinquin

(3 mM in DMSO)

(5 mM in ethanol or DMSO)

I add5 ol/ml

add 10 lal/ml [ cell culture volume I x

J

\

cell culture volume

/ J incubate for 30 / m i n at 37°C / (intracellularesteraseactivitytransforms

incubate for 15 N min at 4~C \

Localization of intracellular Zn2+-release by fluorescence microscopy of live cells or

Quantification of intracellular ZnZ+-release by FACS analysis FIG. 1. Scheme to localize or quantify NO-mediated intracellular Z n 2÷ release,

chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) at a concentration of 200/zM eliminates the presence of free Zn 2+, thus inhibiting Zn 2+ labeling by TSQ or Zinquin. We successfully found intracellular Zn 2+ release after NO treatment in primary murine and human endothelial cells of various origin, in freshly isolated splenocytes, 6 and in several other cell lines tested (murine fibroblasts, human MCF-7 breast adenocarcinomas, rat kangaroo Pt K kidney cells, COS cells). In contrast, no increase in Zn2+-specific fluorescence was found in Chinese hamster ovary (CHO) cells after NO treatment, for reasons we do not know. Freshly isolated hepatocytes contain extremely high concentrations of intracellular labile Zn 2+, rendering these cells extremely

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I lZinquin+ SNOC

[ 131

Zinquin + I

Zinquin fluorescence FIG. 2. Histograms of mouse spleen cells incubated for 1 hr with 25/xM Zinquin in the absence (left) or presence of 10 mM SNOC (middle) or 10 mM SNOC NO (right). Gating of living cells was performed according to light scatter and propidium iodide fluorescence. The SNOC-treated splenocytes are shifted to 100% into the positive log scale, showing that NO mediates intracellular Zn2+-release.

bright after labeling. Treatment with NO did not result in a visible enhancement of this already extremely bright Zn2+-dependent fluorescence.

Quantification of Zn 2÷ Release after Exogenous NO Application ZnZ+-specific increase in intracellular fluorescence intensity induced by NO treatment can be quantified by analysis via fluorescence-activated cell sorting (FACS) using a 530 nm bandpass filter. However, when adherent ceils are used, these have to be detached from the culture dish by treatment with trypsin prior to analysis. With fibroblasts, this results in a strong autofluorescence signal which interferes with the ZnZ+-dependent Zinquin fluorescence. Therefore, we only use nonadherent cells for FACS analysis. Simultaneous staining with propidium iodide (excitation at about 470-550 nm, emission at about 580-650 nm) to mark dead cells allows for detection of live labeled cells only. An example quantitating intracellular ZnZ+-release in live splenocytes is given in Fig. 2. The method for measuring labeled cells by FACS has previously been described in detail. 19

Investigation of NO-Mediated Effects on Zinc Finger Transcription Factors in Ceils To investigate whether NO interacts with zinc finger transcription factors in living cells, these are treated with subtoxic concentrations of NO donors. For studying constitutive transcription with low transcription rates, an NO donor with a long half-life (DETA/NO, SNAP) should be used. In contrast, transcription induced by drugs or other factors should be investi19 D. R. Parks and L. A. Herzenberg, Methods Enzymol. 108, 197 (1984).

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Cell culture ] constitutivetranscription

induced transcription

... t..... l

.....

NO concentration via NO-donor with long half life (eg SNAP, DETA/NO)

h12-24~

NO concentration via NO-donor with short half life (e.g. SNOC, PAPA]NO)

1-2 h

Preparation of nuclear extracts

Gel-shift assay to quantitate specific DNA-binding activity of the respective zinc finger transcription factor FIG. 3. Scheme to investigate effects of NO on zinc finger transcription factors in cells.

gated using an NO donor with a shorter half-life (PAPA/NO, SNOC) (Fig. 3). As NO-mediated effects in cells may be reversible, incubation times with SNOC should not exceed 1-2 hr, and with D E T A / N O should not exceed 12-24 hr. After the end of the incubation, cells are lysed by hypotonic buffer following high salt extraction of nuclear proteins. 2°,21For studying DNA-binding activities of nuclear proteins, gel-shift assays using labeled D N A fragments or oligonucleotide templates are the method of choice as described in detail elsewhere? 2,23 An example of such a gel-shift assay after treatment of lymphocytes with interleukin-1/~ (IL-1/3) and SNOC is given in Fig. 4. We found that treatment of cells with a concentration of 1-2 mM D E T A / NO and 0.5-1 mM SNOC, respectively, is sufficient to inhibit D N A binding of zinc finger transcription factors involved in constitutive and induced 2oN. C. Andrews and D. V. Failer, Nucl. Acids. Res. 11, 2499 (1991). 21 y. j. Suzuki and L. Packer, Methods Enzymol. 252, 175 (1995). 22j. Carey, Methods Enzymol. 208, 103 (1991). 23D. Lane, P. Prentki, and M. Chandler, Microbiol. Rev. 56, 509 (1992).

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a

b

c

d

[ 13]

e

f

FIG. 4. ELA-6.1 lymphocytes were incubated for 3 hr without (lane b) or with 1000 U/ml Interleukin-lfl (lanes e-f) in the absence or presence of 0.5 mM SNOC (lane d) and 1 mM SNOC (lane e), respectively, or 1 mM denitrosylated SNOC (SNOC_No, lane f). Subsequently, nuclear extracts were prepared, and a gel-shift assay was performed using a labeled Splspecific oligonucleotide template. In lane a, recombinant Spl has been loaded on the gel instead of a nuclear extract. As can clearly be seen, treatment of cells with SNOC destroyed the DNA-binding activity of Spl in the nucleus, while the control compound, SNOC_No, had no effect.

t r a n s c r i p t i o n , r e s p e c t i v e l y . P r o o f t h a t N O - m e d i a t e d effects a r e specific for zinc finger t r a n s c r i p t i o n factors is given b y s t u d y i n g t h e D N A - b i n d i n g activity of t r a n s c r i p t i o n factors t h a t d o n o t c o n t a i n zinc fingers o r c y s t e i n e residues. N e i t h e r N F - K B n o r A P - 1 can b e u s e d as controls, as b o t h c o n t a i n a c y s t e i n e i n v o l v e d in t h e D N A - b i n d i n g which can b e i n h i b i t e d b y N O via S-nitrosylation.14 Conclusion N O m e d i a t e s i n t r a c y t o p l a s m i c a n d i n t r a n u c l e a r Z n 2+ r e l e a s e t h a t c a n b e l o c a l i z e d a n d q u a n t i f i e d using Zn2÷-specific f l u o r o p h o r e s . N O - i n d u c e d d e s t r u c t i o n o f zinc finger d o m a i n s in t r a n s c r i p t i o n factors is o n e m e c h a n i s m

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to regulate gene expression, We here have described methods to apply NO to cells following an assay to investigate the DNA-binding activity of transcription factors. This may allow an understanding of NO-mediated gene regulatory effects. The methods described are easy and rapid to perform and may allow for investigating NO-mediated effects on Zn 2+ metabolism, ZnZ+-dependent signal transduction, or transcription.

[ 14] I m m u n o p r e c i p i t a t i o n of NitrotyrosineContaining Proteins

By LEE A N N

M A C M I L L A N - C R o w a n d J O H N A . THOMPSON

Introduction Several mechanisms, all of which involve nitric oxide (NO), have been described which lead to nitration of tyrosine residues in proteins. One mechanism involves the diffusion-limited reaction of NO with superoxide (O~) generating the potent oxidant and nitrating agent peroxynitrite (ONOO). 1,2 At pathophysiological concentrations NO is the only known biological molecule that can outcompete endogenous superoxide dismutase (SOD) for available O~ ,3 and formation of ONOO accounts for both O~ and NO-dependent toxicities. Another oxidant, hypochlorous acid (HOC1, a product of activated neutrophils), in the presence of nitrite (a breakdown product of NO) and a peroxidase can initiate nitrotyrosine formation. 4 The HOCl-mediated nitration would be an extremely effective process in the close vicinity of a neutrophil, whereas the ONOO-mediated process would be capable of nitrating proteins in numerous microenvironments (including the mitochondria). Others have suggested that nitrite and hydrogen peroxide (H202) result in formation of nitrotyrosine; however, this mechanism requires extremely high concentrations of both reactants and an acidic environment. 5 Regardless of these alternative mechanisms, nitration of tyrosine residues in target proteins produces a permanent modification that can be detected immunologically. Nitrotyrosine immunoreactivity has been reported in several human 1 R. E. Huie and S. Padmaja, Free Rad. Res. Commun. 18, 195 (1993). : J. S. Beckman, T. W. Beckman, J. Chen, P. A. Marshall, and B. A. Freeman, Proc. NatL Acad. Sci. USA 87, 1620 (1990). 3 j. S. Beckman and J. P. Crow, Biochem. Soc. Trans. 21, 330 (1993). 4 A. van der Vliet, J. P. Eiserich, B. Halliwell, and C. E. Cross, J. Biol. Chem. 272, 7617 (1997). 5 T. D. Oury, L. Tatro, A. J. Ghio, and C. A. Piantadosi, Free Rad. Res. 23, 537 (1995).

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