Debkumar Pain

Ionizing radiation-induced metabolic oxidative stress and prolonged cell injury

Cellular exposure to ionizing radiation leads to oxidizing events that alter atomic structure through direct interactions of radiation with target macromolecules or via products of water radiolysis. Further, the oxidative damage may spread from the targeted to neighboring, non-targeted bystander cells through redox-modulated intercellular communication mechanisms. To cope with the induced stress and the changes in the redox environment, organisms elicit transient responses at the molecular, cellular and tissue levels to counteract toxic effects of radiation. Metabolic pathways are induced during and shortly after the exposure. Depending on radiation dose, dose-rate and quality, these protective mechanisms may or may not be sufficient to cope with the stress. When the harmful effects exceed those of homeostatic biochemical processes, induced biological changes persist and may be propagated to progeny cells. Physiological levels of reactive oxygen and nitrogen species play critical roles in many cellular functions. In irradiated cells, levels of these reactive species may be increased due to perturbations in oxidative metabolism and chronic inflammatory responses, thereby contributing to the long-term effects of exposure to ionizing radiation on genomic stability. Here, in addition to immediate biological effects of water radiolysis on DNA damage, we also discuss the role of mitochondria in the delayed outcomes of ionization radiation. Defects in mitochondrial functions lead to accelerated aging and numerous pathological conditions. Different types of radiation vary in their linear energy transfer (LET) properties, and we discuss their effects on various aspects of mitochondrial physiology. These include short and long-term in vitro and in vivo effects on mitochondrial DNA, mitochondrial protein import and metabolic and antioxidant enzymes.

Yeast Mitochondrial Protein, Nfs1p, Coordinately Regulates Iron-Sulfur Cluster Proteins, Cellular Iron Uptake, and Iron Distribution

Nfs1p is the yeast homolog of the bacterial proteins NifS and IscS, enzymes that release sulfur from cysteine for iron-sulfur cluster assembly. Here we show that the yeast mitochondrial protein Nfs1p regulates cellular and mitochondrial iron homeostasis. A strain of Saccharomyces cerevisiae, MA14, with a missenseNFS1 allele (I191S) was isolated in a screen for altered iron-dependent gene regulation. This mutant exhibited constitutive up-regulation of the genes of the cellular iron uptake system, mediated through effects on the Aft1p iron-regulatory protein. Iron accumulating in the mutant cells was retained in the mitochondrial matrix while, at the same time, iron-sulfur proteins were deficient. In this work, the yeast protein was localized to mitochondria, and the gene was shown to be essential for viability. Furthermore, Nfs1p in the MA14 mutant was found to be markedly decreased, suggesting that this low protein level produced the observed regulatory effects. This hypothesis was confirmed by experiments in which expression of wild-type Nfs1p from a regulated galactose-induced promoter was turned off, leading to recapitulation of the iron regulatory phenotypes characteristic of the MA14 mutant. These phenotypes include decreases in iron-sulfur protein activities coordinated with increases in cellular iron uptake and iron distribution to mitochondria.

The Mechanism of Action of Steroidogenic Acute Regulatory Protein (StAR) StAR ACTS ON THE OUTSIDE OF MITOCHONDRIA TO STIMULATE STEROIDOGENESIS

Steroidogenic acute regulatory protein (StAR) plays an essential role in steroidogenesis, facilitating delivery of cholesterol to cytochrome P450scc on the inner mitochondrial membrane. StAR is synthesized in the cytoplasm and is subsequently imported by mitochondria and processed to a mature form by cleavage of the NH2-terminal mitochondrial targeting sequence. To explore the mechanism of StAR action, we produced 6-histidine-tagged N-62 StAR (His-tag StAR) constructs lacking the NH2-terminal 62 amino acids that encode the mitochondrial targeting sequence and examined their steroidogenic activity in intact cells and on isolated mitochondria. His-tag StAR proteins stimulated pregnenolone synthesis to the same extent as wild-type StAR when expressed in COS-1 cells transfected with the cholesterol side-chain cleavage system. His-tag StAR was diffusely distributed in the cytoplasm of transfected COS-1 cells whereas wild-type StAR was localized to mitochondria. There was no evidence at the light or electron microscope levels for selective localization of His-tag StAR protein to mitochondrial membranes. In vitro import assays demonstrated that wild-type StAR preprotein was imported and processed to mature protein that was protected from subsequent trypsin treatment. In contrast, His-tag StAR was not imported and protein associated with mitochondria was sensitive to trypsin. Using metabolically labeled COS-1 cells transfected with wild-type or His-tag StAR constructs, we confirmed that wild-type StAR preprotein was imported and processed by mitochondria, whereas His-tag StAR remained largely cytosolic and unprocessed. To determine whether cytosolic factors are required for StAR action, we developed an assay system using washed mitochondria isolated from bovine corpora lutea and purified recombinant His-tag StAR proteins expressed in Escherichia coli. Recombinant His-tag StAR stimulated pregnenolone production in a dose- and time-dependent manner, functioning at nanomolar concentrations. A point mutant of StAR (A218V) that causes lipoid congenital adrenal hyperplasia was incorporated into the His-tag protein. This mutant was steroidogenically inactive in COS-1 cells and on isolated mitochondria. Our observations conclusively document that StAR acts on the outside of mitochondria, independent of mitochondrial import, and in the absence of cytosol. The ability to produce bioactive recombinant StAR protein paves the way for refined structural studies of StAR and StAR mutants.

Mt-Hsp70 Homolog, Ssc2p, Required for Maturation of Yeast Frataxin and Mitochondrial Iron Homeostasis

Here we show that the yeast mitochondrial chaperone Ssc2p, a homolog of mt-Hsp70, plays a critical role in mitochondrial iron homeostasis. Yeast withssc2-1 mutations were identified by a screen for altered iron-dependent gene regulation and mitochondrial dysfunction. These mutants exhibit increased cellular iron uptake, and the iron accumulates exclusively within mitochondria. Yfh1p is homologous to frataxin, the human protein implicated in the neurodegenerative disease, Friedreich’s ataxia. Like mutants ofyfh1, ssc2-1 mutants accumulate vast quantities of iron in mitochondria. Furthermore, using import studies with isolated mitochondria, we demonstrate a specific role for Ssc2p in the maturation of Yfh1p within this organelle. This function for a mitochondrial Hsp70 chaperone is likely to be conserved, implying that a human homolog of Ssc2p may be involved in iron homeostasis and in neurodegenerative disease.

Bimodal targeting of microsomal CYP2E1 to mitochondria through activation of an N-terminal chimeric signal by cAMP-mediated phosphorylation.

Cytochrome P450 2E1 (CYP2E1) plays an important role in alcohol-induced toxicity and oxidative stress. Recently, we showed that this predominantly microsomal protein is also localized in rat hepatic mitochondria. In this report, we show that the N-terminal 30 amino acids of CYP2E1 contain a chimeric signal for bimodal targeting of the apoprotein to endoplasmic reticulum (ER) and mitochondria. We demonstrate that the cryptic mitochondrial targeting signal at sequence 21–31 of the protein is activated by cAMP-dependent phosphorylation at Ser-129. S129A mutation resulted in lower affinity for binding to cytoplasmic Hsp70, mitochondrial translocases (TOM40 and TIM44) and reduced mitochondrial import. S129A mutation, however, did not affect the extent of binding to the signal recognition particle and association with ER membrane translocator protein Sec61. Addition of saturating levels of signal recognition particle caused only a partial inhibition of CYP2E1 translation under in vitro conditions, and saturating levels of ER resulted only in partial membrane integration. cAMP enhanced the mitochondrial CYP2E1 (referred to as P450MT5) level but did not affect its level in the ER. Our results provide new insights on the mechanism of cAMP-mediated activation of a cryptic mitochondrial targeting signal and regulation of P450MT5 targeting to mitochondria.

Dual targeting of cytochrome P4502B1 to endoplasmic reticulum and mitochondria involves a novel signal activation by cyclic AMP‐dependent phosphorylation at Ser128

We have investigated mechanisms of mitochondrial targeting of the phenobarbital‐inducible hepatic mitochondrial P450MT4, which cross‐reacts with antibody to microsomal P4502B1. Results show that P4502B1 and P450MT4 have identical primary sequence but different levels of phosphorylation and secondary structure. We demonstrate that P4502B1 contains a chimeric mitochondrial and endoplasmic reticulum (ER) targeting signal at its N‐terminus. Inducers of cAMP and protein kinase A‐mediated phosphorylation of P4502B1 at Ser128 activate the signal for mitochondrial targeting and modulate its mitochondrial or ER destination. S128A mutation inhibits in vitro mitochondrial transport and also in vivo mitochondrial targeting in COS cells. A fragment of P4502B1 containing the N‐terminal signal and the phosphorylation site could drive the transport of dihydrofolate reductase (DHFR) into mitochondria. Ser128 phosphorylation reduced the affinity of 2B1 protein for binding to SRP, but increased the affinity of the 2B1–DHFR fusion protein for binding to yeast mitochondrial translocase proteins, TOM40 and TIM44, and matrix Hsp70. We describe a novel regulatory mechanism by which cAMP modulates the targeting of a protein to two distinct organelles.

Frataxin and Mitochondrial FeS Cluster Biogenesis

Friedreich ataxia is an inherited neurodegenerative disease caused by frataxin deficiency. Frataxin is a conserved mitochondrial protein that plays a role in FeS cluster assembly in mitochondria. FeS clusters are modular cofactors that perform essential functions throughout the cell. They are synthesized by a multistep and multisubunit mitochondrial machinery that includes the scaffold protein Isu for assembling a protein-bound FeS cluster intermediate. Frataxin interacts with Isu, iron, and the cysteine desulfurase Nfs1, which supplies sulfide, thus placing it at the center of mitochondrial FeS cluster biosynthesis.

Dre2, a Conserved Eukaryotic Fe/S Cluster Protein, Functions in Cytosolic Fe/S Protein Biogenesis

ABSTRACT In a forward genetic screen for interaction with mitochondrial iron carrier proteins in Saccharomyces cerevisiae, a hypomorphic mutation of the essential DRE2 gene was found to confer lethality when combined with Δmrs3 and Δmrs4. The dre2 mutant or Dre2-depleted cells were deficient in cytosolic Fe/S cluster protein activities while maintaining mitochondrial Fe/S clusters. The Dre2 amino acid sequence was evolutionarily conserved, and cysteine motifs (CX2CXC and twin CX2C) in human and yeast proteins were perfectly aligned. The human Dre2 homolog (implicated in blocking apoptosis and called CIAPIN1 or anamorsin) was able to complement the nonviability of a Δdre2 deletion strain. The Dre2 protein with triple hemagglutinin tag was located in the cytoplasm and in the mitochondrial intermembrane space. Yeast Dre2 overexpressed and purified from bacteria was brown and exhibited signature absorption and electron paramagnetic resonance spectra, indicating the presence of both [2Fe-2S] and [4Fe-4S] clusters. Thus, Dre2 is an essential conserved Fe/S cluster protein implicated in extramitochondrial Fe/S cluster assembly, similar to other components of the so-called CIA (cytoplasmic Fe/S cluster assembly) pathway although partially localized to the mitochondrial intermembrane space.

Voltage-gated K+ Channels Contain Multiple Intersubunit Association Sites

A domain in the cytoplasmic NH2 terminus of voltage-gated K+ channels supervises the proper assembly of specific tetrameric channels (Li, M., Jan, J. M., and Jan, L. Y. (1992) Science 257, 1225-1230; Shen, N. V., Chen X., Boyer, M. M., and Pfaffinger, P. (1993) Neuron 11, 67-76). It is referred to as a first tetramerization domain, or T1 (Shen, N. V., Chen X., Boyer, M. M., and Pfaffinger, P. (1993) Neuron 11, 67-76). However, a deletion mutant of Kv1.3 that lacks the first 141 amino acids, Kv1.3 (T1−) forms functional channels, suggesting that additional association sites in the central core of Kv1.3 mediate oligomerization. To characterize these sites, we have tested the abilities of cRNA Kv1.3 (T1−) fragments co-injected with Kv1.3 (T1−) to suppress current in Xenopus oocytes. The fragments include portions of the six putative transmembrane segments, S1 through S6, specifically: S1, S1-S2, S1-S2-S3, S2-S3, S2-S3-S4, S3-S4, S3-S4-S5, S2 through COOH, S3 through COOH, S4 through COOH, and S5-S6-COOH. Electrophysiologic experiments show that the fragments S1-S2-S3, S3-S4-S5, S2 through COOH, and S3 through COOH strongly suppress Kv1.3 (T1−) current, while others do not. Suppression of expressed current is due to specific effects of the translated peptide Kv1.3 fragments, as validated by in vivo immunoprecipitation studies of a strong suppressor and a nonsuppressor. Pulse-chase experiments indicate that translation of truncated peptide fragments neither prevents translation of Kv1.3 (T1−) nor increases its rate of degradation. Co-immunoprecipitation experiments suggest that suppression involves direct association of a peptide fragment with Kv1.3 (T1−). Fragments that strongly suppress Kv1.3 (T1−) also suppress an analogous NH2-terminal deletion mutant of Kv2.1 (Kv2.1 (ΔN139)), an isoform belonging to a different subfamily. Our results indicate that sites in the central core of Kv1.3 facilitate intersubunit association and that there are suppression sites in the central core, which are promiscuous across voltage-gated K+ channel subfamilies.

Preparation of protein A-peroxidase monoconjugate using a heterobifunctional reagent, and its use in enzyme immunoassays

Protein A-peroxidase monoconjugate was prepared in solution using a heterobifunctional reagent, N-succinimidyl-3-(2-pyridyldithio)-propionate. The yield of the monoconjugate was much higher than that obtained with current methods. An immunoassay method was developed in which protein A-peroxidase monoconjugate served as a universal tool. Protein A-peroxidase monoconjugate was taken up by IgG molecules immobilized on an excess of solid phase antigen. The ability of free antigen to inhibit the binding of antibody, measured as inhibition of conjugate up take, served as the basis for quantification in the assay. The method was applied to human chorionic gonadotropin (HCG) and human IgG. Optimal assay conditions were developed and it was found that as little as 1 ng of HCG/ml and 2 ng of IgG/ml could be detected. The method is of comparable sensitivity to other available immunoassay methods and gives accurate, reproducible results.