Epidermal pathogenesis of inflammatory dermatoses.

It is generally assumed that dermatitis, whether of allergic or irritant origin, is primarily an immunological/inflammatory disorder. In this article, we review recent information that supports an epidermal contribution to these disorders, as well as several other dermatoses. We first review new concepts of the epidermal barrier, with recent evidence that the stratum corneum is a biosensor that regulates the epidermal lipid and DNA-metabolic responses to a variety of exogenous insults. Various signaling mechanisms, including changes in levels of epidermal cytokines and growth factors, are potential candidates to mediate these metabolic responses. Our results show that these signaling molecules may be generated not in response to permeability barrier requirements, but as an avoidable consequence of the epidermal injury that accompanies all types of acute barrier abrogation. Although the role of cytokines/growth factors as regulators of metabolic events leading to barrier recovery is still unknown, their role in initiating a cytokine cascade leading to cutaneous pathology seems more certain. We conclude that signaling molecules, released following injury to the stratum corneum, initiate a cytokine cascade that induces inflammation, which is responsible for the clinical features of specific dermatoses. Thus, 'outside-to-inside' signaling may contribute to the pathogenesis of a variety of dermatoses characterized by abnormal barrier function.

Cloning of murine translation initiation factor 6 and functional analysis of the homologous sequence YPR016c in Saccharomyces cerevisiae.

The cDNA sequence of a murine gene whose expression was up-regulated after epidermal injury was cloned utilizing differential display. The full-length cDNA was isolated by 3' and 5' rapid amplification of cDNA ends from mouse liver. The predicted protein is >97% identical to the human sequence for eukaryotic translation initiation factor (eIF) 6, thus identifying the gene as murine eIF6. Functional studies of the yeast eIF6 homolog, YPR016c, were initiated in Saccharomyces cerevisiae to determine the cellular role(s) of eIF6. Complete deletion of the YPR016c coding sequence was lethal. Viability was restored in the presence of either YPR016c or murine eIF6, when either was expressed as amino-terminal green fluorescent protein fusion protein. Moreover, both fusion proteins localized to nuclear/perinuclear compartments in their respective yeast strains. When the expression of YPR016c-green fluorescent protein was repressed, there was a dramatic reduction in the 60 S ribosomal subunit and polysome content and decreased 80S monosome content. Additionally, the YPR016c-depleted cells arrested in G1. These studies show that YPR016c, which encodes yeast eIF6, is necessary for maximal polysome formation and plays an important role in determining free 60 S ribosomal subunit content.

Support groups for patients with Graves' disease and other thyroid conditions.

Thyroid foundations and associations provide free information and support for thyroid patients in countries throughout the world. Their efforts save physicians time and increase patient understanding and compliance while reducing anxiety. Personal responsiveness to a patient's concerns is important but costly. Physicians can help by referring patients to local groups, accepting patient referrals from these organizations, and encouraging thyroid patients and organizations to help with financial support.

Barrier disruption increases gene expression of cytokines and the 55 kD TNF receptor in murine skin.

The signalling mechanisms that regulate epidermal permeability barrier homeostasis are not known. Previous Northern blot analysis showed that both acute and chronic barrier disruption increase mRNA levels of several cytokines in murine epidermis. To further characterize the epidermal response to barrier abrogation, we used more sensitive, multi-probe RNase protection assays to measure the mRNA levels of additional cytokines, as well as cytokine receptors in acute and chronic models of barrier disruption. Normal mouse epidermis expressed interleukin (IL)-1 alpha, interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) and IL-6 mRNAs. Following tape-stripping, only the mRNA levels for TNF-alpha, IL-1 alpha, IL-1 beta and IL-6 increased at 2.5 and 7 h, and returned toward normal levels by 18 h. No mRNAs encoding TNF-beta, IL-2, IL-3, IL-4 or IL-5, were detected in the epidermis either under basal conditions or after tape-stripping. Similarly, in a chronic model, essential fatty acid deficiency, epidermal levels of TNF-alpha, IL-1 alpha, IL-1 beta and IL-6 mRNAs, but not IFN-gamma mRNA, were elevated over controls; and again, mRNAs for the remaining probed cytokines were not detected. In contrast, in the dermis, only IL-1 beta mRNA levels increased 2.5 h after tape-stripping, and remained elevated at 18 h. mRNAs encoding the IL-1 (p60), IFN-gamma and IL-6 receptors were present in epidermis, but their levels remained unchanged following either acute or chronic barrier disruption. In contrast, epidermal TNF (p55) receptor mRNA levels were increased by 87% (P < 0.01) at 2.5 h, returned to control levels at 7 h and were increased by 68% (P < 0.03) at 18 h after tape-stripping. The increase at 2 h was confirmed by Northern blot analysis and was not prevented by latex occlusion performed immediately after tape-stripping mRNAs for the IL-1 (p80) receptor and TNF (p75) receptor were not detected in epidermis. Low levels of TNF (p55) receptor mRNA were present in the dermis, and they remained unchanged after tape-stripping. The presence of specific receptor mRNAs in the epidermis and dermis suggests that these tissues are capable of responding in an autocrine and/or paracrine fashion to the cognate cytokines. These results suggest that epidermal cytokines produced after barrier disruption may initiate a cytokine cascade which could regulate cytokine and cytokine receptor production and/or inflammatory responses.

Amphiregulin and nerve growth factor expression are regulated by barrier status in murine epidermis.

Disruption of the murine permeability barrier by solvents or tape stripping stimulates a homeostatic repair response that includes increased epidermal DNA synthesis. To identify potential mediators of the increase in DNA synthesis, we have measured epidermal levels of mRNAs encoding various growth factors after acute barrier disruption. In this study, mRNAs for amphiregulin and nerve growth factor were each shown to increase over controls at 30 min, reach peak levels of 12- to 30-fold at 1-2 h, and return to control levels by 6 h after tape stripping. A similar time course for the increase of amphiregulin and nerve growth factor mRNAs was observed after an unrelated form of barrier disruption, i.e., acetone treatment. Furthermore, artificial restoration of the barrier by Latex occlusion, immediately following barrier disruption by acetone treatment, inhibited the increase in epidermal amphiregulin and nerve growth factor mRNA levels, indicating that barrier status regulates the production of these growth factors. In contrast, mRNA levels of transforming growth factor-beta1, an inhibitory growth factor, were unchanged at early times and decreased by 53% (p < 0.02) 6 h after tape stripping, whereas mRNA levels of transforming growth factor-alpha remained unchanged at all times after acute barrier disruption. These results suggest that barrier disruption stimulates the expression of amphiregulin and nerve growth factor. Together, these regulators of keratinocyte growth and differentiation may be responsible for the increased proliferative response that is associated with barrier disruption.

Barrier disruption stimulates interleukin-1 alpha expression and release from a pre-formed pool in murine epidermis.

Previous studies have shown that barrier disruption increases epidermal mRNA levels of interleukin-1 alpha (IL-1 alpha). We used immunohistochemistry to examine IL-1 alpha expression in hairless mouse skin under basal conditions and following barrier abrogation. In untreated mice, IL-1 alpha was present in the dermis and nucleated epidermal layers in a diffuse, generalized pattern. In essential fatty acid deficient mice IL-1 alpha was present in all epidermal layers and the dermis, with prominent staining in the stratum corneum. After acute barrier disruption with tape-stripping, IL-1 alpha increased in the epidermis and dermis within 10 min, remained elevated at 2 and 4 h, and decreased to near basal levels by 24 h. Moreover, intense, perinuclear, basal cell staining appeared at 10 min, persisting until 4 h after barrier disruption. Since the increase in IL-1 alpha immunostaining after acute barrier abrogation precedes the increase in mRNA, we hypothesized that the IL-1 alpha might derive from a pre-formed pool. Prolonged occlusion of normal skin, a treatment that specifically reduces epidermal mRNA levels of IL-1 alpha, decreased basal immunostaining for IL-1 alpha and blunted the increase in IL-1 alpha usually seen following barrier disruption. Moreover, tape-stripping of skin, maintained ex vivo at 4 degrees C, resulted in increased IL-1 alpha immunostaining within the upper nucleated epidermal layers, as well as release of mature IL-1 alpha into the medium, as measured by Western blotting and enzyme-linked immunosorbent assay. In addition, the stratum corneum attached to the tape contained IL-1 alpha. These studies show that acute barrier disruption induces both the immediate release and dispersion of IL-1 alpha from a pre-formed, epidermal pool, as well as increased IL-1 alpha synthesis; both mechanisms are consistent with a role for IL-1 alpha in the regulation of proinflammatory and homeostatic processes in the skin.

Cutaneous permeability barrier disruption increases fatty acid synthetic enzyme activity in the epidermis of hairless mice.

Previous studies have shown that abrogation of the cutaneous permeability barrier stimulates epidermal fatty acid synthesis and that this increase is required for barrier repair. The purpose of the present study was to determine the enzymatic basis for this increase in synthesis. Acute barrier disruption by tape stripping increased both acetyl CoA carboxylase (62%) and fatty acid synthase (54%) activities in the epidermis. Similarly, acute disruption of the barrier by topical acetone treatment increased epidermal acetyl CoA carboxylase (69%) and fatty acid synthase (43%) activities. In both acute models, provision of an artificial barrier by occlusion with an impermeable membrane prevented the increase in acetyl CoA carboxylase and fatty acid synthase activities, indicating that the increased activity was dependent on an increase in transepidermal water loss and cannot be attributed to nonspecific effects. In addition, chronic disruption of the barrier, produced by feeding an essential-fatty-acid-deficient diet, also increased acetyl CoA carboxylase (127%) and fatty acid synthase (49%) activities in the epidermis. Again, occlusion with an impermeable membrane decreased both acetyl CoA carboxylase and fatty acid synthase activities toward normal. These results indicate that the increase in fatty acid synthesis that occurs in the epidermis after barrier disruption is due to a coordinate increase in the activities of both epidermal acetyl CoA carboxylase and fatty acid synthase.

Occlusion lowers cytokine mRNA levels in essential fatty acid-deficient and normal mouse epidermis, but not after acute barrier disruption.

Acute disruption of the permeability barrier by either tape stripping or acetone treatment and chronic disruption by feeding an essential fatty acid-deficient diet increase the mRNA levels of tumor necrosis factor-alpha (TNF alpha), interleukin (IL)-1 alpha, IL-1 beta, IL-1ra, and granulocyte/macrophage-colony-stimulating factor in murine epidermis. Furthermore, epidermal TNF alpha protein levels also are stimulated by barrier disruption. To understand the relation of epidermal cytokine production to barrier function, we studied the effect of the application of a water vapor-impermeable membrane on epidermal cytokine production both in normal epidermis and after barrier disruption. Latex occlusion of essential fatty acid-deficient mice for 24-48 h lowered the mRNA levels of epidermal TNF alpha, IL-1 alpha, and IL-1ra to nearly control values, but not the levels of IL-1 beta mRNA. Occlusion of normal mice for 8, 24, and 48 h did not alter the levels of epidermal mRNAs encoding TNF alpha, IL-1 beta, or IL-1ra. Yet mRNA levels of IL-1 alpha, the major constitutively produced epidermal cytokine, were reduced by 40% after 24 h and by 80% after 48 h of occlusion of normal mouse epidermis. In contrast, latex occlusion of mice immediately after acute barrier disruption by either tape stripping or acetone treatment blocked neither the stimulation of epidermal mRNAs for TNF alpha, IL-1 alpha, IL-1 beta, or IL-1ra, nor the increase in epidermal TNF alpha protein. Taken together, these results suggest that barrier status regulates the production of specific cytokines in essential fatty acid-deficient and normal mouse epidermis. However, the signals that regulate epidermal cytokine production in response to acute barrier disruption do not appear to be influenced by occlusion.

Barrier function coordinately regulates epidermal IL-1 and IL-1 receptor antagonist mRNA levels.

Disruption of the cutaneous permeability barrier increases mRNA levels for TNF, GM-CSF, IL-1 alpha, and IL-1 beta in the epidermis. We have hypothesized that the cytokines mediate the changes in lipid and DNA synthesis which occur following barrier disruption. To further characterize the cytokine response to barrier abrogation, we examined the levels of epidermal IL-1ra mRNA in two acute models and one chronic model in the hairless mouse. IL-1ra mRNA levels increased shortly after acute disruption of the barrier with acetone, reached a peak at 3-4 h after treatment, and returned to control levels by 8h. These changes in mRNA levels parallel those which occur for IL-1 alpha and beta. Furthermore, IL-1ra mRNA levels were elevated 5-fold and 4-fold, at 2.5 h and 4 h, respectively, following tape-stripping, a second acute model of barrier disruption. Finally, IL-1ra mRNA levels were elevated 2.5-fold in the epidermis of EFAD mice, which have a chronic barrier defect. Thus, the cutaneous response to barrier disruption includes mechanisms which increase IL-1 and IL-1ra mRNA levels in a coordinate manner. The net result provides a regulatory mechanism for controlling the biological effects of increased IL-1 production.

Diabetes increases hepatic hydroxymethyl glutaryl coenzyme A reductase protein and mRNA levels in the small intestine.

Previous studies have shown that both cholesterol synthesis and the activity of hepatic hydroxymethyl glutaryl coenzyme A (HMG CoA) reductase, the rate-limiting enzyme in cholesterol synthesis, are increased in the small intestine of a wide variety of different animal models of diabetes. In the present study, we demonstrate that the mass of HMG CoA reductase protein is increased in the small intestine of both streptozocin-induced diabetic rats (2.5-fold) and streptozocin/alloxan-induced diabetic dogs (2.4-fold). These increases in HMG CoA reductase protein mass are of a magnitude similar to the previously observed increases in either HMG CoA reductase activity and/or cholesterol synthesis in the small intestine of diabetic animals. Furthermore, mRNA levels for HMG CoA reductase in the small intestine of diabetic rats and diabetic dogs are increased 2.1- and 1.7-fold, respectively. These results suggest that the increase in HMG CoA reductase protein levels in the small intestine of diabetic animals is due to an increase in mRNA levels. In contrast, mRNA levels for HMG CoA reductase in the liver of diabetic rats are not increased. Additionally, mRNA levels for the low-density lipoprotein (LDL) receptor are also increased in the small intestine of diabetic animals (rats, 43%; dogs, 59%). The increase in small-intestinal cholesterol synthesis has the potential for adversely affecting lipoprotein metabolism and increasing the risk of atherosclerosis in diabetes.

Permeability barrier disruption alters the localization and expression of TNF alpha/protein in the epidermis.

Previous studies have shown that (1) epidermal TNF alpha mRNA levels are increased following acute disruption of the cutaneous permeability barrier; (2) this increase is maximal at 1 h and decreases to control levels by 8 h; and (3) in essential fatty acid-deficient (EFAD) mice, a chronic model of barrier perturbation, TNF alpha mRNA levels are also elevated several-fold over controls. In the present study we determined, using immunocytochemical procedures, epidermal TNF alpha protein levels following either acute of chronic barrier disruption and the localization of any increase. Frozen, paraffin and Antibed sections of skin were incubated with polyclonal anti-mouse TNF alpha antisera and detection was accomplished by either immunoperoxidase or fluorescence procedures. We found that (1) TNF alpha-immunoreactive protein was present in normal mouse epidermis, and was primarily localized to the upper nucleated layers where it displayed a diffuse cytosolic pattern; (2) acute disruption of the barrier with acetone or tape-stripping resulted in TNF alpha staining that was more intense throughout all of the nucleated epidermal cell layers in comparison with normal epidermis; (3) the increase in TNF alpha staining occurred as early as 2 h after barrier disruption; and (4) increased TNF alpha staining was also observed in the stratum corneum of EFAD mice. These results indicate that epidermal TNF alpha protein levels increase after both acute and chronic barrier disruption, and are consistent with the hypothesis that TNF alpha may signal and/or coordinate portions of the cutaneous response to barrier disruption.

Effect of cutaneous permeability barrier disruption on HMG-CoA reductase, LDL receptor, and apolipoprotein E mRNA levels in the epidermis of hairless mice.

Disruption of the permeability barrier results in an increase in cholesterol synthesis in the epidermis. Inhibition of cholesterol synthesis impairs the repair and maintenance of barrier function. The increase in epidermal cholesterol synthesis after barrier disruption is due to an increase in the activity of epidermal HMG-CoA (3-hydroxy-3-methylglutaryl CoA) reductase. To determine the mechanism for this increase in enzyme activity, in the present study we have shown by Western blot analysis that there is a 1.5-fold increase in the mass of HMG-CoA reductase after acute disruption of the barrier with acetone. In a chronic model of barrier disruption, essential fatty acid deficiency, there is a 3-fold increase in the mass of HMG-CoA reductase. Northern blot analysis demonstrated that after acute barrier disruption with acetone or tape-stripping, epidermal HMG-CoA reductase mRNA levels are increased. In essential fatty acid deficiency, epidermal HMG-CoA reductase mRNA levels are increased 3-fold. Thus, both acute and chronic barrier disruption result in increases in epidermal HMG-CoA reductase mRNA levels which could account for the increase in HMG-CoA reductase mass and activity. Additionally, both acute and chronic barrier disruption increase the number of low density lipoprotein (LDL) receptors and LDL receptor mRNA levels in the epidermis. Moreover, epidermal apolipoprotein E mRNA levels are increased by both acute and chronic perturbations in the barrier. Increases in these proteins in response to barrier disruption may allow for increased lipid synthesis and transport between cells and facilitate barrier repair.

Cutaneous barrier perturbation stimulates cytokine production in the epidermis of mice.

The disruption of the cutaneous permeability barrier results in metabolic events that ultimately restore barrier function. These include increased epidermal sterol, fatty acid, and sphingolipid synthesis, as well as increased epidermal DNA synthesis. Because tumor necrosis factor (TNF) and other cytokines are known products of keratinocytes and have been shown to modulate lipid and DNA synthesis in other systems, their levels were examined in two acute models and one chronic model of barrier perturbation in hairless mice. Acute barrier disruption with acetone results in a 72% increase in epidermal TNF 2.5 h after treatment, as determined by Western blotting. Furthermore, epidermal TNF mRNA was elevated ninefold over controls 2.5 h after acetone treatment. This elevation in TNF mRNA was maximal at 1 h after acetone, and decreased to control levels by 8 h. After tape stripping, a second acute model of barrier disruption that avoids application of potentially toxic chemicals, TNF mRNA was elevated fivefold over controls at 2.5 h. Moreover, the mRNA levels for epidermal IL-1 alpha, IL-1 beta, and granulocyte macrophage-colony-stimulating factor (GM-CSF) also were elevated several-fold over controls, after either acetone treatment or tape stripping, but their kinetics differed. GM-CSF mRNA reached a maximal level at 1 h after acetone, while IL-1 alpha and IL-1 beta were maximal at 4 h after treatment. In contrast, mRNAs encoding IL-6 and IFN gamma were not detected either in control murine epidermis or in samples obtained at various times after tape stripping or acetone treatment. The relationship of the cytokine response to barrier function is further strengthened by results obtained in essential fatty acid deficient mice. In this chronic model of barrier perturbation mRNA levels for epidermal TNF, IL-1 alpha, IL-1 beta, and GM-CSF were each elevated several-fold over controls. These results suggest that epidermal cytokine production is increased after barrier disruption and may play a role in restoring the cutaneous permeability barrier.

Autoimmune thyroid disease, left-handedness, and developmental dyslexia.

Autoimmune thyroid disease (ATD) (Graves' disease and Hashimoto's thyroiditis) has been one of a number of autoimmune diseases possibly associated with left-handedness, mixed dominance, and learning disability. In the present study, 74 men with ATD were compared to 24 control men with non-ATD. An increased frequency of mixed dominance and traits suggestive of dyslexia were observed in the patients with ATD. These data are consistent with a link between the development of ATD and cerebral dominance.

A third mutation at the CpG dinucleotide of codon 504 and a silent mutation at codon 506 of the HEX A gene.

Two CpG mutations at codon 504 of the gene encoding the alpha-subunit of beta-hexosaminidase (the HEX A gene) have been identified previously: (1) a C deletion resulting in premature termination of the alpha-subunit and (2) a G----A transition resulting in 504Arg----His substitution, in patients with infantile Tay-Sachs disease and juvenile GM2 gangliosidosis, respectively. This prompted a search for a C----T transition in the same dinucleotide, as would be expected from the mechanism of CpG mutagenesis. Such a mutation, which results in a substitution of cysteine for arginine, was found in a patient with chronic GM2 gangliosidosis, in compound heterozygosity with the known 269Gly----Ser allele. The biochemical phenotype of the 504Arg----Cys mutation was examined by site-directed mutagenesis of the alpha-subunit cDNA and transfection of Cos-1 cells. The expression of the mutagenized cDNA with the cysteine substitution gave rise to an alpha-subunit with the same defects as those resulting from expression of mutagenized cDNA with the histidine substitution--i.e., secretion primarily as the alpha-monomer rather than as the alpha alpha dimer, along with absence of enzymatic activity. The 504Arg----Cys/269Gly----Ser genotype of the chronic GM2 gangliosidosis patient is shared by her sibling, who has mild adult-onset GM2 gangliosidosis, implying that the clinical differences between them must be attributed to other factors. The family is unique in yet another respect--namely, that the normal allele of the mother and of a 504Arg----Cys heterozygous sibling has a silent mutation, a G----A transition in the wobble position of the glutamic acid codon at position 506.(ABSTRACT TRUNCATED AT 250 WORDS)

A cystic fibrosis phenotype in cells cultured from sweat gland secretory coil. Altered kinetics of 36Cl efflux.

As a step toward understanding the metabolic consequences of the cystic fibrosis (CF) mutation, we have examined the kinetics of 36Cl efflux in cells cultured from sweat glands, a tissue that is affected in the disease. Epithelial cells, derived from the secretory coil of sweat glands of CF and control individuals, were cultured in serum-free medium, and primary cultures used for efflux experiments. Cell layers were equilibrated with Na36Cl in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-buffered balanced salt solution for 45 min at 37 degrees C, washed in 0.25 M sucrose, and incubated in nonradioactive buffer for measurement of 36Cl efflux. Efflux from CF and control cells followed biphasic kinetics and was described by the equation Y = Ae-kat + Be-kbt. All efflux was inhibited at 6 degrees C. The fast component of efflux, Ae-kat, of both control and CF cells was inhibited by the anion channel blockers 4,4'-diisothiocyanato-2,2'-stilbene disulfonic acid, 9-anthracene carboxylate, and diphenylamine 2-carboxylate, implicating release through chloride channels. At 23 degrees C, the kinetics of 36Cl efflux from CF and control cells were indistinguishable, but efflux from control cells could be accelerated by cAMP analogs and isoproterenol. At 37 degrees C, 36Cl efflux was more rapid from control cells than from CF cells, but could not be stimulated further by beta-adrenergic agents. In both cases, the increased rate of efflux was due to a severalfold increase in the A parameter of the fast component. These differential responses constitute a "CF phenotype" of secretory sweat gland cells in culture that may be useful for further investigation of the metabolic defect in cystic fibrosis.

Replacement of a conserved proline and the alkaline conformational change in iso-2-cytochrome c.

Although point mutations usually lead to minor localized changes in protein structure, replacement of conserved Pro-76 with Gly in iso-2-cytochrome c induces a major conformational change. The change in structure results from mutation-induced depression of the pK for transition to an alkaline conformation with altered heme ligation. To assess the importance of position 76 in stabilizing the native versus the alkaline structure, the equilibrium and kinetic properties of the pH-induced conformational change have been compared for normal and mutant iso-2-cytochrome c. The pKapp for the conformational change is reduced from 8.45 (normal iso-2) to 6.71 in the mutant protein (Gly-76 iso-2), suggesting that conservation of Pro-76 may be required to stabilize the native conformation at physiological pH. The kinetics of the conformational change for both the normal and mutant proteins are well-described by a single kinetic phase throughout most of the pH-induced transition zone. Over this pH range, a minimal mechanism proposed for horse cytochrome c [Davis, L. A., Schejter, A., & Hess, G. P. (1974) J. Biol. Chem. 249, 2624-2632] is consistent with the data for normal and mutant yeast iso-2-cytochromes c: NH KH----N + H+ kcf in equilibrium kcb A NH and N are native forms of cytochrome c with a 695-nm absorbance band, A is an alkaline form that lacks the 695-nm band, KH is a proton dissociation constant, and kcf and kcb are microscopic rate constants for the conformational change. The Gly-76 mutation increases kcf by almost 70-fold, but kcb and KH are unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

Replacement of a conserved proline eliminates the absorbance-detected slow folding phase of iso-2-cytochrome c.

As a test of the proline isomerization model, we have used oligonucleotide site-directed mutagenesis to construct a mutant form of iso-2-cytochrome c in which proline-76 is replaced by glycine [Wood, L. C., Muthukrishnan, K., White, T. B., Ramdas, L., & Nall, B. T. (1988) Biochemistry (preceding paper in this issue)]. For the oxidized form of Gly-76 iso-2, an estimate of stability by guanidine hydrochloride induced unfolding indicates that the mutation destabilizes the protein by 1.2 kcal/mol under standard conditions of neutral pH and 20 degrees C (delta G degrees u = 3.8 kcal/mol for normal Pro-76 iso-2 versus 2.6 kcal/mol for Gly-76 iso-2). The kinetics of folding/unfolding have been monitored by fluorescence changes throughout the transition region using stopped-flow mixing. The rates for fast and slow fluorescence-detected refolding are unchanged, while fast unfolding is increased in rate 3-fold in the mutant protein compared to normal iso-2. A new kinetic phase in the 1-s time range is observed in fluorescence-detected unfolding of the mutant protein. The presence of the new phase is correlated with the presence of species with an altered folded conformation in the initial conditions, suggesting assignment of the phase to unfolding of this species. The fluorescence-detected and absorbance-detected slow folding phases have been monitored as a function of final pH by manual mixing between pH 5.5 and 8 (0.3 M guanidine hydrochloride, 20 degrees C).(ABSTRACT TRUNCATED AT 250 WORDS)

Construction and characterization of mutant iso-2-cytochromes c with replacement of conserved prolines.

Oligonucleotide-directed mutagenesis has been used to construct two mutant forms of iso-2-cytochrome c. In one, Pro-30 is replaced by threonine; in the other, Pro-76 is replaced by glycine. Both prolines are fully conserved among mitochondrial cytochromes c and play important structural and functional roles. Yeast with either the Pro-30 or the Gly-76 mutation has appreciable levels of mutant protein in vivo and grows on media containing nonfermentable carbon sources. Thus, neither mutation blocks protein targeting to mitochondria, uptake by mitochondria, covalent attachment of heme, or in vivo function. As judged by ultraviolet-visible spectrophotometry and proton nuclear magnetic resonance spectroscopy, the nativelike conformation of purified Gly-76 iso-2 at pH 6 is almost indistinguishable from that of the normal protein at pH 6. Ultraviolet second-derivative spectrophotometry, however, suggests an increase in the average number of exposed tyrosine side chains, with 2.25 out of 5 residues exposed for the mutant compared to 1.95 for normal iso-2. Above neutral pH, the protein folds to a mutant conformation possibly related to alkaline cytochrome c. Nuclear Overhauser difference spectroscopy of the reduced nativelike conformation allows assignment of several proton resonances and comparison of side-chain conformations of the heme ligand Met-80 in the mutant and the normal proteins. The proton chemical shifts for the assigned resonances are the same within errors for Gly-76 iso-2 and normal iso-2 at pD 6, 20 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)