Nuclear envelope assembly defects link mitotic errors to chromothripsis.

Defects in the architecture or integrity of the nuclear envelope are associated with a variety of human diseases1. Micronuclei, one common nuclear aberration, are an origin for chromothripsis2, a catastrophic mutational process that is commonly observed in cancer3-5. Chromothripsis occurs after micronuclei spontaneously lose nuclear envelope integrity, which generates chromosome fragmentation6. Disruption of the nuclear envelope exposes DNA to the cytoplasm and initiates innate immune proinflammatory signalling7. Despite its importance, the basis of the fragility of the micronucleus nuclear envelope  is not known. Here we show that micronuclei undergo defective nuclear envelope assembly. Only 'core' nuclear envelope proteins8,9 assemble efficiently on lagging chromosomes, whereas 'non-core' nuclear envelope proteins8,9, including nuclear pore complexes (NPCs), do not. Consequently, micronuclei fail to properly import key proteins that are necessary for the integrity of the nuclear envelope and genome. We show that spindle microtubules block assembly of NPCs and other non-core nuclear envelope proteins on lagging chromosomes, causing an irreversible defect in nuclear envelope assembly. Accordingly, experimental manipulations that position missegregated chromosomes away from the spindle correct defective nuclear envelope assembly, prevent spontaneous nuclear envelope disruption, and suppress DNA damage in micronuclei. Thus, during mitotic exit in metazoan cells, chromosome segregation and nuclear envelope assembly are only loosely coordinated by the timing of mitotic spindle disassembly. The absence of precise checkpoint controls may explain why errors during mitotic exit are frequent and often trigger catastrophic genome rearrangements4,5.

A peroxidase mimetic protects skeletal muscle cells from peroxide challenge and stimulates insulin signaling.

Reactive oxygen species such as hydrogen peroxide have been implicated in causing metabolic dysfunction such as insulin resistance. Heme groups, either by themselves or when incorporated into proteins, have been shown to scavenge peroxide and demonstrate protective effects in various cell types. Thus, we hypothesized that a metalloporphyrin similar in structure to heme, Fe(III)tetrakis(4-benzoic acid)porphyrin (FeTBAP), would be a peroxidase mimetic that could defend cells against oxidative stress. After demonstrating that FeTBAP has peroxidase activity with reduced nicotinamide adenine dinucleotide phosphate (NADPH) and NADH as reducing substrates, we determined that FeTBAP partially rescued C2C12 myotubes from peroxide-induced insulin resistance as measured by phosphorylation of AKT (S473) and insulin receptor substrate 1 (IRS-1, Y612). Furthermore, we found that FeTBAP stimulates insulin signaling in myotubes and mouse soleus skeletal muscle to about the same level as insulin for phosphorylation of AKT, IRS-1, and glycogen synthase kinase 3ß (S9). We found that FeTBAP lowers intracellular peroxide levels and protects against carbonyl formation in myotubes exposed to peroxide. Additionally, we found that FeTBAP stimulates glucose transport in myotubes and skeletal muscle to about the same level as insulin. We conclude that a peroxidase mimetic can blunt peroxide-induced insulin resistance and also stimulate insulin signaling and glucose transport, suggesting a possible role of peroxidase activity in regulation of insulin signaling.

Reversible Oxidative Modifications in Myoglobin and Functional Implications.

Myoglobin (Mb), an oxygen-binding heme protein highly expressed in heart and skeletal muscle, has been shown to undergo oxidative modifications on both an inter- and intramolecular level when exposed to hydrogen peroxide (H2O2) in vitro. Here, we show that exposure to H2O2 increases the peroxidase activity of Mb. Reaction of Mb with H2O2 causes covalent binding of heme to the Mb protein (Mb-X), corresponding to an increase in peroxidase activity when ascorbic acid is the reducing co-substrate. Treatment of H2O2-reacted Mb with ascorbic acid reverses the Mb-X crosslink. Reaction with H2O2 causes Mb to form dimers, trimers, and larger molecular weight Mb aggregates, and treatment with ascorbic acid regenerates Mb monomers. Reaction of Mb with H2O2 causes formation of dityrosine crosslinks, though the labile nature of the crosslinks broken by treatment with ascorbic acid suggests that the reversible aggregation of Mb is mediated by crosslinks other than dityrosine. Disappearance of a peptide containing a tryptophan residue when Mb is treated with H2O2 and the peptide's reappearance after subsequent treatment with ascorbic acid suggest that tryptophan side chains might participate in the labile crosslinking. Taken together, these data suggest that while exposure to H2O2 causes Mb-X formation, increases Mb peroxidase activity, and causes Mb aggregation, these oxidative modifications are reversible by treatment with ascorbic acid. A caveat is that future studies should demonstrate that these and other in vitro findings regarding properties of Mb have relevance in the intracellular milieu, especially in regard to actual concentrations of metMb, H2O2, and ascorbate that would be found in vivo.

Myoglobin as a versatile peroxidase: Implications for a more important role for vertebrate striated muscle in antioxidant defense.

Myoglobins (Mb) are ubiquitous proteins found in striated muscle of nearly all vertebrate taxa. Although their function is most commonly associated with facilitating oxygen storage and diffusion, Mb has also been implicated in cellular antioxidant defense. The oxidized (Fe3+) form of Mb (metMB) can react with hydrogen peroxide (H2O2) to produce ferrylMb. FerrylMb can be reduced back to metMb for another round of reaction with H2O2. In the present study, we have shown that horse skeletal muscle Mb displays peroxidase activity using 2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) and 3,3',5,5'-tetramethylbenzidine (TMB) as reducing substrates, as well as the biologically-relevant substrates NADH/NADPH, ascorbate, caffeic acid, and resveratrol. We have also shown that ferrylMb can be reduced by both ethanol and acetaldehyde, which are known to accumulate in some vertebrate tissues under anaerobic conditions, such as anoxic goldfish and crucian carp, implying a potential mechanism for ethanol detoxification in striated muscle. We found that metMb peroxidase activity is pH-dependent, increasing as pH decreases from 7.4 to 6.1, which is biologically relevant to anaerobic vertebrate muscle when incurring intracellular lactic acidosis. Finally, we found that metMb reacts with hypochlorite in a heme-dependent fashion, indicating that Mb could play a role in hypochlorite detoxification. Taken together, these data suggest that Mb peroxidase activity might be an important antioxidant mechanism in vertebrate cardiac and skeletal muscle under a variety of physiological conditions, such as those that might occur in contracting skeletal muscle or during hypoxia.

The paradoxical role of IL-10 in immunity and cancer.

Interleukin-10 (IL-10) produced by a wide-variety of cells is a highly pleiotropic cytokine. It has been implicated in the pathogenesis and/or development of autoimmune diseases and cancer, although it displays differential effects that seem to be contradictory sometimes. The ultimate role of this cytokine in disease, however, cannot be fully determined until the immunological contexts that regulate its function are further elucidated. In this review, we will discuss a wide variety of evidence of IL-10 in immunity and cancer in an effort to illuminate the remaining mysteries in the function of this cytokine that, when fully understood, may prove to be a powerful tool in the battle against cancer.