Calponin is a troponin-T like protein purified from chicken gizzard smooth muscle. It binds to actin, myosin, Ca(2+)-binding proteins and tropomyosin and inhibits the actomyosin ATPase as well as the movement of actin filaments over myosin in vitro. These properties have led to the proposal that calponin may be involved in the Ca(2+)-dependent regulation of actin-myosin interaction and consequently of smooth muscle contraction. Calponin is localized in both the contractile and the cytoskeletal parts of the smooth muscle cell and may have a structural function in smooth muscle cells. It may also regulate the pool of free actin available for cytoskeleton organization. In vitro calponin function is modulated by its interaction with a Ca(2+)-binding protein and/or by its phosphorylation. This suggests that calponin may play an important role in signal transduction from the membrane receptor to the contractile proteins in smooth muscle.
Pyruvate carboxylase purified from chicken liver shows an essentially absolute requirement for activation by an acyl coenzyme A in all preparations and under all conditions tested thus far. The enzyme is activated by formyl-, acetyl-, propionyl-, crotonyl-, and iso-butyryl-CoA, but carboxylated analogues of the activating acyl derivatives of CoA and certain other acetyl thioesters are potent competitive inhibitors with respect to acetyl-CoA. Examination of the initial reaction rate as a function of activator concentration reveals a sigmoid relationship, suggesting that more than 1 molecule of activator per active site is required for the activation process or that cooperative interactions between the bound activator molecules occur. Inactivation analyses show an interaction of acetyl-CoA with pyruvate carboxylase, with resultant marked alterations in the tertiary and quaternary structures of the protein as compared with the free enzyme. For example, the rate of inactivation of this biotin enzyme by avidin is markedly increased in the presence of acetyl-CoA concentrations similar to those required for activation of the over-all reaction. Acetyl-CoA also affords almost complete protection of this enzyme against denaturation at 2° or by low concentrations of urea and sodium dodecyl sulfate. These findings are consistent with the proposal that acetyl-CoA and the other activator acyl-CoA derivatives act as allosteric effectors for pyruvate carboxylase. However, acetyl-CoA in the presence or absence of other reaction components has no significant effect either on the sedimentation properties of pyruvate carboxylase in the range from 0.5 to 5.0 mg of protein per ml or on the absorption spectrum of this enzyme. Pyruvate carboxylase catalyzes a very slow hydrolysis of acyl thioesters. An apparent relationship between thioester hydrolysis and the activation of the over-all reaction is suggested by studies of the specificity of hydrolysis and of the effects of other reaction components on the rate of hydrolysis. The evidence appears to exclude the possibility that the activated species of pyruvate carboxylase is an acyl-enzyme, and the observed thioester hydrolysis may therefore be related indirectly to the activation process.
Studies in unicellular organisms have revealed a complex role of superoxide dismutase (SOD) in aerobic cell metabolism. A compromise in SOD activity can have broad ramifications on cell function, ranging from defects in amino acid metabolism to increases in oxidative damage to DNA. From the studies on organisms genetically defective for SOD, it appears that at least three basic functions can be assigned to this ubiquitous metal-binding enzyme: (1) SODs protect biomolecules against the damage from O2−; (2) Oxidation by O2− of iron-sulfur clusters becomes problematic in cells lacking SOD; and (3) damage to these clusters can inactivate key enzymes involved in amino acid and sugar metabolism and also lead to a mobilization of free iron and an increase in iron-related toxicity. It is noteworthy that from studies in yeast, all the cellular damage resulting from loss of SOD can be reversed by alterations in the homeostasis of the transition metals copper and manganese. Currently, it remains unclear as to whether these metals are functionally substituting for SOD at the level of O2¯ scavenging, metal buffering, or maintaining the reduced environment of the cell.
In contrast to the conventional notion that reactive oxygen is mostly a trigger for oxidative damage of biological structures, now we know that low physiologically relevant concentrations of ROS can regulate a variety of key molecular mechanisms that may be linked with important cell functions (Fig. 4). Redox-based regulation of gene expression has emerged as a fundamental regulatory mechanism in cell biology. Several proteins, with apparent redox-sensing activity, have been described. Electron flow through side-chain functional CH2-SH groups of conserved cysteinyl residues in these proteins account for the redox-sensing properties. Protein thiol groups with high thiol-disulfide oxidation potentials are likely to be redox-sensitive. The ubiquitous endogenous thiols thioredoxin and glutathione are of central importance in redox signaling. Signals are transduced from the cell surface to the nucleus through phosphorylation and dephosphorylation chain reactions of cellular proteins at tyrosine and serine/threonine. Protein phosphorylation, one of the most fundamental mediators of cell signaling, is redox-sensitive. DNA-binding proteins are involved in the regulation of cellular processes such as replication, recombination, viral integration and transcription. Several studies show that the interaction of certain transcription regulatory proteins with their respective cognate DNA sites is also redox-regulated. Changes in the concentration of Ca2+i control a wide variety of cellular functions, including transcription and gene expression; Ca(2+)-driven protein phosphorylation and proteolytic processing of proteins are two major intracellular events that are implicated in signal transduction from the cell surface to the nucleus. Intracellular calcium homeostasis is regulated by the redox state of cellular thiols, and it is evident that cell calcium may play a critical role in the activation of the redox-sensitive transcription factor NF-kappa B. Among the several thiol agents tested for their efficacy in modulating cellular redox status, N-acetyl-L-cysteine and alpha-lipoic acid hold most promise for human use. A strong therapeutic potential of strategies that would modulate the cellular thioredoxin system has been also evident.
Publisher Summary To maintain balanced metabolism, Escherichia coli must coordinate the assimilation of nitrogen with the assimilation of carbon and other essential nutrients. This coordination is accomplished in part by a signal transduction system that measures the signals of carbon and nitrogen status and regulates the activity of glutamine synthetase (GS) and the transcription of nitrogen-regulated (Ntr) genes, whose products facilitate the use of poor nitrogen sources. The key sensory components of this signal transduction system are the uridylyltransferase/ uridylyl-removing enzyme (UTase/UR), PII protein, and adenylyltransferase (ATase) that regulates GS by reversible adenylylation. This chapter discusses the current state of understanding of these signal-transducing proteins and the mechanisms by which they detect and transduce the signals of nitrogen and carbon status. It also discusses the physiology of the response to nitrogen and carbon availability and presents an overview of the signal transduction system.
The activity of NF-kappa B is critically involved in the inflammatory activation of endothelial cells and their adhesiveness and also appears to regulate apoptosis in SMC by coordinating antiapoptotic programs. The activity of NF-kappa B has been revealed within human atheromas or following angioplasty but not in undiseased arteries. Hence, the inhibition of NF-kappa B mobilization by antioxidative or anti-inflammatory agents or by adenoviral I kappa B alpha overexpression, as reviewed herein, may act in concert to suppress endothelial activation and to induce SMC apoptosis. This synergistic concept may be a vasoprotective approach to prevent atherogenesis and restenosis by attenuating inflammatory reactions and SMC proliferation in nascent and progressing atherosclerotic lesions, as well as in developing neointima formations following angioplasty.
The HO-1 isoenzyme is an early stress response gene regulated by many forms of oxidative stress. The HO-2 isoenzyme is predominantly a constitutive enzyme, which may serve to sequester heme as well as degrade it. All HO enzyme activity results in the degradation of heme and the production of antioxidant bile pigments, which would favor an antioxidant role for the enzyme. In fact, in oxidative stress in vitro, HO-1 is protective (91-94) but within a narrow threshold of overexpression (93,94) in some models, since iron released in the HO reaction may obviate any cytoprotective effect (Fig. 3). So far, HO-2 appears to be beneficial in oxygen toxicity in vivo, but the consequences of HO-2 overexpression have not yet been tested. It will be important to better define the role of each HO isoenzyme in oxidative stress so as to determine whether enhancing these complex systems could alleviate some of the cellular changes seen as a result of oxidative injury. Furthermore, prior to considering therapeutic maneuvers to enhance HO, a complete understanding of the physiologic consequences of HO-1 induction and associated reactions, in each particular setting, will be crucial.
The thiol redox status of intracellular and extracellular compartments is critical in the determination of protein structure, regulation of enzyme activity, and control of transcription factor activity and binding. Thiol antioxidants act through a variety of mechanisms, including (1) as components of the general thiol/disulfide redox buffer, (2) as metal chelators, (3) as radical quenchers, (4) as substrates for specific redox reactions (GSH), and (5) as specific reductants of individual protein disulfate bonds (thioredoxin). The composition and redox status of the available thiols in a given compartment is highly variable and must play a part in determining the metabolic activity of each compartment. It is generally beneficial to increase the availability of specific antioxidants under conditions of oxidant stress. Cells have devised a number of mechanisms to promote increased intracellular levels of thiols such as GSH and thioredoxin in response to a wide variety of stresses. Exogenous thiols have been used successfully to increase cell and tissue thiol levels in cell cultures, in animal models, and in humans. Increased levels of GSH and other thiols have been associated with increased tolerance to oxidant stresses in all of these systems and in some cases, with disease prevention or treatment in humans. A wide variety of thiol-related compounds have been used for these purposes. These include thiols such as GSH and its derivatives, cysteine and NAC, dithiols such as lipoic acid, which is reduced to the thiol form intracellularly, and "prothiol" compounds such as OTC, which are enzymatically converted to free thiols within the cell. In choosing a thiol for a specific function (e.g., protection of lung from oxidant exposure or protection of organs from ischemia reperfusion injury), the global effects must also be considered. For example, large increases in free thiols in the circulation are associated with toxic effects. These effects may be the result of thiyl radical-mediated reactions but could also be due to destabilizing effects of increases in thiol/disulfide ratios in the plasma, which normally is in a more oxidized state than intracellular compartments. Changes in the thiol redox gradient across cells could also adversely affect any transport or cell signaling processes, which are dependent on formation and rupture of disulfide linkages in membrane proteins. Therapeutic thiol administration has been shown to have great potential, and its efficacy should be increased by selecting compounds and methods of delivery that will minimize perturbations in the thiol status of regions external to the targeted areas.
Glutathione (GSH) is a ubiquitous intracellular peptide with diverse functions that include detoxification, antioxidant defense, maintenance of thiol status, and modulation of cell proliferation. GSH is synthesized in the cytosol of all mammalian cells in a tightly regulated manner. The major determinants of GSH synthesis are the availability of cysteine, the sulfur amino acid precursor, and the activity of the rate-limiting enzyme, glutamate cysteine ligase (GCL). GCL is composed for a catalytic (GCLC) and modifier (GCLM) subunit and they are regulated at multiple levels and at times differentially. The second enzyme of GSH synthesis, GSH synthase (GS) is also regulated in a coordinated manner as GCL subunits and its up-regulation can further enhance the capacity of the cell to synthesize GSH. Oxidative stress is well known to induce the expression of GSH synthetic enzymes. Key transcription factors identified thus far include Nrf2/Nrf1 via the antioxidant response element (ARE), activator protein-1 (AP-1) and nuclear factor κ B (NFκB). Dysregulation of GSH synthesis is increasingly being recognized as contributing to the pathogenesis of many pathological conditions. These include diabetes mellitus, pulmonary fibrosis, cholestatic liver injury, endotoxemia and drug-resistant tumor cells. Manipulation of the GSH synthetic capacity is an important target in the treatment of many of these disorders.
Antioxidants are substances that delay or prevent the oxidation of cellular oxidizable substrates. The various antioxidants exert their effect by scavenging superoxide or by activating a battery of detoxifying/defensive proteins. In this chapter, we have focused on the mechanisms by which antioxidants induce gene expression. Many xenobiotics (e.g., beta-naphthoflavone) activate genes similar to those activated by antioxidants. The promoters of these genes contain a common cis-element, termed the antioxidant response element (ARE), which contains two TRE (TPA response element) or TRE-like elements followed by GC box. Mutational studies have identified GTGAC***GC as the core of the ARE sequence. Many transcription factors, including Nrf, Jun, Fos, Fra, Maf, YABP, ARE-BP1, Ah (aromatic hydrocarbon) receptor, and estrogen receptor bind to the ARE from the various genes. Among these factors, Nrf-Jun heterodimers positively regulate ARE-mediated expression and induction of genes in response to antioxidants and xenobiotics. This Nrf-Jun heterodimerization and binding to the ARE requires unknown cytosolic factors. The mechanism of signal transduction from antioxidants and xenobiotics includes several steps: (1) Antioxidants and xenobiotics undergo metabolism to generate superoxide and related reactive species, leading to the generation of a signal to activate expression of detoxifying/defensive genes. (2) The generation of superoxide and related reactive species is followed by activation of yet to be identified cytosolic factors by unknown mechanism(s). (3). Activated cytosolic factors catalyze modification of Nrf and/or Jun proteins, which bind to the ARE in promoters of the various detoxifying/defensive genes. (4) The transcription of genes encoding detoxifying/defensive proteins is increased. The unknown cytosolic factors are significant molecules because they represent the oxidative sensors within the cells. Identification of the cytosolic factors will be of considerable importance in the field of antioxidants and gene regulation research. Future studies will also be required to completely understand the molecular mechanism of signal transduction from antioxidants and xenobiotics to Nrf-Jun. In addition to the Nrf-Jun pathway, mammalian cells also contain other pathways that activate gene expression in response to oxidative stress. These include NF-KB-, HIF-1-, Mac-1-, and SRF-mediated pathways. It is expected that collectively these pathways increase transcription of more than four dozen genes to protect cells against oxidative stress.
Using immunohistochemistry, Fas and caspase 8 positive sites were localized in the brains of Alzheimer patients and those with multiple infarcts as well as age-matched normal. The frontal, occipital and hippocampal regions were dissected out from these patients and normal individuals shortly after death. Positive Fas neurons were found in all these regions of normal individuals and Alzheimer patients, but not in the brains of patients with multiple infarct. Activated caspase 8 protein was found only in the brains of Alzheimer patients and not in the other groups. Plaques in the Alzheimer patients were positive for Fas and activated caspase 8. The former was localized in the membrane and the inside of the plaque whereas activated caspase 8 was only present in the interior of the plaque, as revealed by confocal microscopy. Our study shows that caspase 8 was activated in the Alzheimer brain and the presence of Fas and activated caspase 8 points to a possibility that at least some plaques were of intracellular origin as Fas was a cell membrane receptor and caspase 8 was a intracellular enzyme downstream to Fas. Furthermore, we had confirmed that in most neurons Fas and caspase 8 did not exist concomitantly and that the glial cells (astrocytes) did not express caspase 8 in Alzheimer patients.
When Lockshin and Zakeri discussed the relevance of apoptosis to aging, the common view was that apoptosis had primarily a negative impact on aging by destroying essential and often irreplaceable cells. That view has now changed to one that acknowledges that there are two general ways in which apoptosis can play a role in aging: (1) elimination of damaged and presumably dysfunctional cells (e.g., fibroblasts, hepatocytes) which can then be replaced by cell proliferation, thereby maintaining homeostasis and elimination of essential postmitotic cells (e.g., neurons) which cannot be replaced, thereby leading to pathology. Evidence exists in two systems (fibroblasts and thymocytes/lymphocytes) that there are age-related decreases in the potential for apoptosis, although the molecular bases for these decreases appear to differ (Table II). Fibroblasts (and neurons?) lose the ability to downregulate bcl-2 in response to an apoptotic signal; thus, apoptosis is blocked even though an initiating signal has been received. In contrast, thymocytes/lymphocytes lack the ability to initiate the signal due to downregulation of the cell surface receptor Fas. There is limited information available for other tissue types, and nothing is known about why and how these age-related changes occur. An interesting observation, but not necessarily a critical one, is that the frequency of upregulation of the bcl-2 gene due to chromosome translocation increases with age. The role of apoptosis in regulating cell number is also a promising area of research. The studies on liver damage and neoplastic lesions suggest an extremely important role for apoptosis in controlling cancer. This may be particularly important in the prostate, where hypertrophy and cancer are a virtual certainty with ever-increasing age. It is not known whether the ability to undergo apoptosis declines in the prostate with increasing age, but it appears likely that it does. One problem in answering questions about the actual regulation of apoptosis is the lack of a quantitative assay. Apoptosis appears to be either "on" or "off" in cells, while the basic cell-killing machinery may often be present, but in an inactive form. Most assays for apoptosis are microscopic rather than kinetic, and the rate-limiting step may be at the level of the initiating signal. Thus, if CR, which extends the life span of rodents, does upregulate apoptosis, it is not clear how to quantify the magnitude of this effect or what should be quantified. The best one can do is to measure the frequency of occurrence of apoptotic bodies. This is essentially a pool size assay which provides little knowledge about how rapidly cells are leaving and entering the pool. Nevertheless, the results currently available do suggest that apoptosis is a process which may be important in aging, at least in some tissues, and the mechanism of its regulation needs to be understood. Although a variety of tumor suppressor gene and oncogene products are known to be involved in signal transduction associated with apoptosis, it remains to be shown which of these, if any, are actually involved in "on-off" switches for apoptosis and which might regulate the intrinsic rate of apoptosis. As Driscoll has already pointed out: "regulation and execution of cell death is an absolutely critical process that interfaces with nearly every aspect of life. Future investigation of the links of cell death to cellular aging and the aging of organisms should be an exciting enterprise."
The secretion mechanisms of cathepsin L from osteoclasts in the process of bone resorption were investigated. The increases in bone pit numbers formed take place by PTH addition in parallel with the increases of cathepsin L and/or L-like proteinase activities in the culture medium of bone cells, and these were suppressed by the addition ofcalcitonin. The Z-Phe-Arg-MCA hydrolysing activity increased in the medium through the effect of PTH is considered to be a kind of procathepsin L by Western blotting analysis, and was suppressed by calcitonin addition. Furthermore, monensin inhibited not only the PTH-induced pit formation, but also cysteine proteinase activity in osteoclasts. Therefore, the procathepsin L excreted might be transferred from endothelial reticulum via Golgi and/or via lysosomes.
We have identified novel signal transduction cascades in activating NF-kappa B, as well as its pathogenetic roles in various disease processes. By applying the basic knowledge obtained through these studies, we hope to find new therapeutic measures against currently incurable diseases such as hematogenic cancer cell metastasis, rheumatoid arthritis, and AIDS. We also propose a novel strategy in screening effective inhibitors against transcription factors. Elucidation of the cis-regulatory element for expression of pathogenetic genes and identification of the responsible transcription factor will not only facilitate the study of pathogenesis but will also promote the development of effective therapy. Recognition of control mechanisms of the NF-kappa B activation pathway has explained the therapeutic efficacy of various compounds with different pharmacologic actions. A similar strategy may be applicable for other inducible transcription factors. From the medical point of view, one of the purposes of these approaches is to find small molecular weight compounds that can be administered orally and that are effective in controlling gene expression of pathogenetic genes.
Iron metabolism is regulated in cells to ensure that iron supplies are adequate and nontoxic. The expression of iron metabolism is regulated primarily by posttranscriptional mechanisms. Ferritin, eALAS, SDHb of Drosophila, and mammalian mitochondrial aconitase are translationally regulated. The TfR is regulated at the level of mRNA stability. Iron regulatory proteins are regulated either by assembly or by disassembly of an iron-sulfur cluster (IRP1) or by rapid degradation in the presence of iron (IRP2). The list of targets for IRP-mediated regulation is growing longer, and a range of possibilities for versatile regulation exists, as each IRP can bind to unique targets that differ from the consensus IRE. The reactivity of iron with oxygen and the creation of toxic by-products may be the evolutionary stimulus that produced this system of tight posttranscriptional gene regulation.
Facultative bacteria can grow under either oxic or anoxic conditions. While oxygen provides substantial advantages in energy yield by respiration, it can become life-threatening because of reactive oxygen species that derive from the molecule naturally. Thus, to survive and thrive in a given niche, these bacteria have to constantly regulate physiological processes to make maximum benefits from oxygen respiration while restraining oxidative stress. Molecular mechanisms and physiological consequences of oxidative stress have been under extensive investigation for decades, mostly on research model Escherichia coli, from which our understanding of bacterial oxidative stress response is largely derived. Nevertheless, given that bacteria live in enormously diverse environments, to cope with oxidative stress different strategies are conceivably developed.
