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Rodney L Levine

Publications and source records attributed to Rodney L Levine.

30 records · Page 2Linked to original sources
PubMed2003

Iron regulatory protein 2 as iron sensor. Iron-dependent oxidative modification of cysteine.

Iron regulatory protein 2 coordinates cellular regulation of iron metabolism by binding to iron responsive elements in mRNA. The protein is synthesized constitutively but is rapidly degraded when iron stores are replete. This iron-dependent degradation requires the presence of a 73-residue degradation domain, but its functions have not yet been established. We now show that the domain can act as an iron sensor, mediating its own covalent modification. The domain forms an iron-binding site with three cysteine residues located in the middle of the domain. It then reacts with molecular oxygen to generate a reactive oxidizing species at the iron-binding site. One cysteine residue is oxidized to dehydrocysteine and other products. This covalent modification may thus mark the protein molecule for degradation by the proteasome system.

Amino Acids↗
PubMed2003

Oxidation of methionine residues of proteins: biological consequences.

Most reactive oxygen species (ROS) can oxidize methionine (Met) residues of proteins to methionine sulfoxide (MetO). However, unlike the ROS-dependent oxidation of other amino acid residues of proteins (except cysteine residues), the oxidation of Met residues is readily reversed by the action of methionine sulfoxide reductase (Msr) that catalyzes the thioredoxin-dependent reduction of MetO residues of proteins back to Met. We summarize here results of studies showing that the cyclic interconversion of Met and MetO residues of proteins is involved in several different biological processes: (a) It is the basis of an important antioxidant mechanism for the scavenging of ROS. (b) It is likely involved in the regulation of enzyme activities. (c) It is involved in cell signaling. (d) It can target proteins for proteolytic degradation. Furthermore, a loss in the ability to catalyze the reduction of protein MetO to Met residues leads to a decrease in the maximum life span, whereas overexpression of this activity leads to an increase in the life span of animals. In addition, a decrease in Msr activities in brain tissues is associated with the development of Alzheimer's disease.

Aging↗
PubMed2002

Direct detection of potential selenium delivery proteins by using an Escherichia coli strain unable to incorporate selenium from selenite into proteins.

Selenium can be metabolized for protein synthesis by two major pathways in vivo. In a specific pathway it can be inserted into polypeptide chains as the amino acid selenocysteine, as directed by the UGA codon. Alternatively, selenium can be substituted for sulfur to generate the free amino acids selenocysteine and selenomethionine, and these are incorporated nonspecifically into proteins in place of cysteine and methionine, respectively. A mutant strain of Escherichia coli was constructed that is deficient in utilization of inorganic selenium for both specific and nonspecific pathways of selenoprotein synthesis. Disruption of the cysK gene prevented synthesis of free cysteine and selenocysteine from inorganic S and Se precursors. Inactivation of the selD gene prevented synthesis of selenophosphate, the reactive selenium donor, required for the specific incorporation pathway. As expected, the double mutant strain, RL165 Delta selD, when grown anaerobically in LB + glucose medium containing (75)SeO(3)(2-), failed to synthesize selenium-dependent formate dehydrogenase H and seleno-tRNAs. However, it incorporated 24% as much selenium as the wild-type strain. Selenium in the deficient strain was bound to five different proteins. A 39-kDa species was identified as glyceraldehyde-3-phosphate dehydrogenase. It is possible that selenium was bound as a perselenide derivative to the reactive cysteine residue of this enzyme. A 28-kDa protein identified as deoxyribose phosphate aldolase also contained bound selenium. These (75)Se-labeled proteins may have alternate roles as selenium delivery proteins.

Aldehyde-Lyases↗
PubMed2002

ADP ribosylation of human neutrophil peptide-1 regulates its biological properties.

In human airways, epithelial cells lining the lumen and intraluminal cells (e.g., polymorphonuclear cells) participate in the innate immune response. These cells secrete or express on their surfaces arginine-specific ADP ribosyltransferases. Defensins, antimicrobial proteins secreted by immune cells, are arginine-rich, leading us to hypothesize that ADP ribosylation could modify their biological activities. We found that an arginine-specific ADP ribosyltransferase-1 present on airway epithelial cells modifies Arg-14 of alpha defensin-1. ADP-ribosylated defensin-1 had decreased antimicrobial and cytotoxic activities but still stimulated T cell chemotaxis and IL-8 release from A549 cells. Further, ADP-ribosylated defensin-1 inhibited cytotoxic and antimicrobial activities of unmodified defensin-1. We identified ADP-ribosylated defensin-1 in bronchoalveolar lavage fluid from smokers but not from nonsmokers, confirming its existence in vivo. Thus, airway mono-ADP-ribosyltransferases could have an important regulatory role in the innate immune response through modification of alpha defensin-1 and perhaps other basic molecules, with alteration of their biological properties.

ADP Ribose Transferases↗
PubMed2002

Carbonyl modified proteins in cellular regulation, aging, and disease.

The oxidative modification of proteins by reactive species is implicated in the etiology or progression of a panoply of disorders and diseases. The level of these modified molecules can be quantitated by measurement of the protein carbonyl content, which has been shown to increase in a variety of diseases and processes, notably during aging. For the most part, oxidatively modified proteins are not repaired and must be removed by proteolytic degradation, a process which normally proceeds very efficiently, from microorganisms to mammals. In eukaryotes, removal is usually carried out by the proteosome, which selectively degrades oxidatively modified proteins, whether they be damaged by reactive oxygen species or specifically oxidized by cellular regulatory processes. The molecular deficiencies that cause accumulation of oxidatively modified proteins are not identified, but regardless of cause, the accumulation is likely to disrupt normal cellular function.

Aging↗
PubMed2002

Assessment of skin carbonyl content as a noninvasive measure of biological age.

The oxidative modification of proteins by reactive species, especially reactive oxygen species, is implicated in the etiology or progression of a panoply of disorders and diseases. For the most part, oxidatively modified proteins are not repaired and must be removed by proteolytic degradation. The level of these modified molecules can be quantitated by measurement of the protein carbonyl content, which has been shown to increase in a variety of diseases and processes, most notably during aging. However, these studies have required invasive techniques to obtain cells for analysis. We examined the possibility that desquamating skin cells (corneocytes) would also show an age-related increase in protein carbonyl content, thus providing a noninvasive method for assessing biological age. This was not the case, as we found no age-dependent relationship in the protein carbonyl content of skin cells from volunteers aged 20 to 79 years.

Adult↗
PubMed2002

Numerous proteins in Mammalian cells are prone to iron-dependent oxidation and proteasomal degradation.

The mechanisms that underlie iron toxicity in cells and organisms are poorly understood. Previous studies of regulation of the cytosolic iron sensor, iron-regulatory protein 2 (IRP2), indicate that iron-dependent oxidation triggers ubiquitination and proteasomal degradation of IRP2. To determine if oxidization by iron is involved in degradation of other proteins, we have used a carbonyl assay to identify oxidized proteins in lysates from RD4 cells treated with either an iron source or iron chelator. Protein lysates from iron-loaded or iron-depleted cells were resolved on two-dimensional gels and these iron manipulations were also repeated in the presence of proteasomal inhibitors. Eleven abundant proteins were identified as prone to iron-dependent oxidation and subsequent proteasomal degradation. These proteins included two putative iron-binding proteins, hNFU1 and calreticulin; two proteins involved in metabolism of hydrogen peroxide, peroxiredoxin 2 and superoxide dismutase 1; and several proteins identified in inclusions in neurodegenerative diseases, including HSP27, UCHL1, actin and tropomyosin. Our results indicate that cells can recognize and selectively eliminate iron-dependently oxidized proteins, but unlike IRP2, levels of these proteins do not significantly decrease in iron-treated cells. As iron overload is a feature of many human neurological diseases, further characterization of the process of degradation of iron-dependently oxidized proteins may yield insights into mechanisms of human disease.

Acetylcysteine↗
PubMedDate not supplied

The role of endogenous heme synthesis and degradation domain cysteines in cellular iron-dependent degradation of IRP2.

Iron regulatory protein 2 (IRP2) is a mammalian cytosolic iron-sensing protein that regulates expression of iron metabolism proteins, including ferritin and transferrin receptor 1. IRP2 is ubiquitinated and degraded by the proteasome in iron-replete cells but is relatively stable in iron-depleted cells. Recent work has shown that IRP2 contains a unique 73-amino-acid domain that binds iron in vitro and undergoes iron-dependent oxidation and cleavage (J. Biol. Chem. 278 (2003), 14857). Several cysteines in the 73-amino-acid domain function as an in vitro iron-binding site. To assess the role of these cysteines in cellular iron- dependent degradation of IRP2, we mutagenized these cysteines in various combinations in the context of full-length protein and generated cell lines in which recombinant IRP2 expression was inducible. Iron-dependent degradation of IRP2 mutagenized at any or all of the cysteines of the putative degradation domain in cells was comparable to wild-type (WT). Both WT and cysteine mutant protein were stabilized in 3% oxygen. Treatment with sodium nitroprusside (SNP), an NO+ donor, caused a decrease in cellular IRP2 concentrations, but the SNP effect was abrogated by simultaneous addition of the iron chelator desferal and was not affected by cysteine mutations. Inhibition of endogenous heme synthesis with succinylacetone significantly inhibited iron- dependent degradation of IRP2. Addition of cobalt chloride inhibited degradation of both WT and mutagenized IRP2. Thus, we could not discern a role for the recently defined in vitro cysteine-dependent iron-binding site of IRP2 in cellular physiology. The early molecular events in iron-dependent degradation of IRP2 remain to be elucidated.

Cysteine↗
PubMedDate not supplied

Molecular determinants of S-glutathionylation of carbonic anhydrase 3.

Carbonic anhydrase 3 is easily S-glutathionylated in vivo and in vitro. The protein has two surface-exposed cysteine residues that can be modified. We found that Cys186 is more readily glutathionylated than Cys181. We studied a series of site-specific mutants to identify the residues that interact with Cys186 to make its thiol more reactive. We found that Lys211 is responsible for lowering the pKa of Cys186. We also found that two acidic residues, Asp188 and Glu212, interact with the thiol and actually decrease its reactivity. We speculate that conformational changes that alter the interaction with these three residues provide a mechanistic basis for modulation of the susceptibility of carbonic anhydrase 3 to glutathionylation.

Alkylation↗
PubMedDate not supplied

Cyclic oxidation and reduction of protein methionine residues is an important antioxidant mechanism.

Almost all forms of reactive oxygen species (ROS) oxidize methionine residues of proteins to a mixture of the R- and S-isomers of methionine sulfoxide. Because organisms contain methionine sulfoxide reductases (Msr's) that can catalyze the thioredoxin-dependent reduction of the sulfoxides back to methionine, it was proposed that the cyclic oxidation/reduction of methionine residues might serve as antioxidants to scavenge ROS, and also to facilitate the regulation of critical enzyme activities. We summarize here results of studies showing that organisms possess two different forms of Msr--namely, MsrA that catalyzes reduction of the S-isomer and MsrB that catalyzes the reduction of the R-isomer. Deletion of the msrA gene in mice leads to increased sensitivity to oxidative stress and to a decrease (40%) in the maximum lifespan. This suggests that elimination of both Msr's would have more serious consequences.

Animals↗