Phytoferritin and its implications for human health and nutrition

Background

Plant and animal ferritins stem from a common ancestor, but plant ferritins exhibit various features that are different from those of animal ferritins. Phytoferritin is observed in plastids (e.g., chloroplasts in leaves, amyloplasts in tubers and seeds), whereas animal ferritin is largely found in the cytoplasm. The main difference in structure between plant and animal ferritins is the two specific domains (TP and EP) at the N-terminal sequence of phytoferritin, which endow phytoferritin with specific iron chemistry. As a member of the nonheme iron group of dietary iron sources, phytoferritin consists of 24 subunits that assemble into a spherical shell storing up to ∼ 2000 Fe^3 + in the form of an iron oxyhydroxide-phosphate mineral. This feature is distinct from small molecule nonheme iron existing in cereals, which has poor bioavailability.

Scope of review

This review focuses on the relationship between structure and function of phytoferritin and the recent progress in the use of phytoferritin as iron supplement.

Major conclusions

Phytoferritin, especially from legume seeds, represents a novel alternative dietary iron source.

General significance

An understanding of the chemistry and biology of phytoferritin, its interaction with iron, and its stability against gastric digestion is beneficial to design diets that will be used for treatment of global iron deficiency.     

Oxido-reduction is not the only mechanism allowing ions to traverse the ferritin protein shell

Background

Most models for ferritin iron release are based on reduction and chelation of iron. However, newer models showing direct Fe(III) chelation from ferritin have been proposed. Fe(III) chelation reactions are facilitated by gated pores that regulate the opening and closing of the channels.

Scope of review

Results suggest that iron core reduction releases hydroxide and phosphate ions that exit the ferritin interior to compensate for the negative charge of the incoming electrons. Additionally, chloride ions are pumped into ferritin during the reduction process as part of a charge balance reaction. The mechanism of anion import or export is not known but is a natural process because phosphate is a native component of the iron mineral core and non-native anions have been incorporated into ferritin in vitro. Anion transfer across the ferritin protein shell conflicts with spin probe studies showing that anions are not easily incorporated into ferritin. To accommodate both of these observations, ferritin must possess a mechanism that selects specific anions for transport into or out of ferritin. Recently, a gated pore mechanism to open the 3-fold channels was proposed and might explain how anions and chelators can penetrate the protein shell for binding or for direct chelation of iron.

Conclusions and general significance

These proposed mechanisms are used to evaluate three in vivo iron release models based on (1) equilibrium between ferritin iron and cytosolic iron, (2) iron release by degradation of ferritin in the lysosome, and (3) metallo-chaperone mediated iron release from ferritin.     

Iron release from ferritin by flavin nucleotides

Background

Extensive in-vitro studies have focused on elucidating the mechanism of iron uptake and mineral core formation in ferritin. However, despite a plethora of studies attempting to characterize iron release under different experimental conditions, the in-vivo mobilization of iron from ferritin remains poorly understood.Several iron-reductive mobilization pathways have been proposed including, among others, flavin mononucleotides, ascorbate, glutathione, dithionite, and polyphenols. Here, we investigate the kinetics of iron release from ferritin by reduced flavin nucleotide, FMNH2, and discuss the physiological significance of this process in-vivo.

Methods

Iron release from horse spleen ferritin and recombinant human heteropolymer ferritin was followed by the change in optical density of the Fe(II)–bipyridine complex using a Cary 50 Bio UV–Vis spectrophotometer. Oxygen consumption curves were followed on a MI 730 Clark oxygen microelectrode.

Results

The reductive mobilization of iron from ferritin by the nonenzymatic FMN/NAD(P)H system is extremely slow in the presence of oxygen and might involve superoxide radicals, but not FMNH2. Under anaerobic conditions, a very rapid phase of iron mobilization by FMNH2 was observed.

Conclusions

Under normoxic conditions, FMNH2 alone might not be a physiologically significant contributor to iron release from ferritin.

General significance

There is no consensus on which iron release pathway is predominantly responsible for iron mobilization from ferritin under cellular conditions. While reduced flavin mononucleotide (FMNH2) is one likely candidate for in-vivo ferritin iron removal, its significance is confounded by the rapid oxidation of the latter by molecular oxygen.     

Serum ferritin: Past,present and future

Background

Serum ferritin was discovered in the 1930s, and was developed as a clinical test in the 1970s. Many diseases are associated with iron overload or iron deficiency. Serum ferritin is widely used in diagnosing and monitoring these diseases.

Scope of review

In this chapter, we discuss the role of serum ferritin in physiological and pathological processes and its use as a clinical tool.

Major conclusions

Although many aspects of the fundamental biology of serum ferritin remain surprisingly unclear, a growing number of roles have been attributed to extracellular ferritin, including newly described roles in iron delivery, angiogenesis, inflammation, immunity, signaling and cancer.

General significance

Serum ferritin remains a clinically useful tool. Further studies on the biology of this protein may provide new biological insights.     

The iron redox and hydrolysis chemistry of the ferritins

Background

Ferritins are ubiquitous and well-characterized iron storage and detoxification proteins. In bacteria and plants, ferritins are homopolymers composed of H-type subunits, while in vertebrates, they typically consist of 24 similar subunits of two types, H and L. The H-subunit is responsible for the rapid oxidation of Fe(II) to Fe(III) at a dinuclear center, whereas the L-subunit appears to help iron clearance from the ferroxidase center of the H-subunit and support iron nucleation and mineralization.

Scope of review

Despite their overall similar structures, ferritins from different origins markedly differ in their iron binding, oxidation, detoxification, and mineralization properties. This chapter provides a brief overview of the structure and function of ferritin, reviews our current knowledge of the process of iron uptake and mineral core formation, and highlights the similarities and differences of the iron oxidation and hydrolysis chemistry in a number of ferritins including those from archaea, bacteria, amphibians, and animals.

General Significance

Prokaryotic ferritins and ferritin-like proteins (Dps) appear to preferentially use H₂O₂ over O₂ as the iron oxidant during ferritin core formation. While the product of iron oxidation at the ferroxidase centers of these and other ferritins is labile and is retained inside the protein cavity, the iron complex in the di-iron cofactor proteins is stable and remains at the catalytic site. Differences in the identity and affinity of the ferroxidase center ligands to iron have been suggested to influence the distinct reaction pathways in ferritins and the di-iron cofactor enzymes.

Major conclusions

The ferritin 3-fold channels are shown to be flexible structures that allow the entry and exit of different ions and molecules through the protein shell. The H- and L-subunits are shown to have complementary roles in iron oxidation and mineralization, and hydrogen peroxide appears to be a by-product of oxygen reduction at the FC of most ferritins. The di-iron(III) complex at the FC of some ferritins acts as a stable cofactor during iron oxidation rather than a catalytic center where Fe(II) is oxidized at the FC followed by its translocation to the protein cavity.     

The extension peptide of plant ferritin from sea lettuce contributes to shell stability and surface hydrophobicity

Plant ferritins have some unique structural and functional features. Most of these features can be related to the plant-specific "extension peptide" (EP), which exists in the N-terminus of the mature region of a plant ferritin. Recent crystallographic analysis of a plant ferritin revealed the structure of the EP, however, two points remain unclear: (i) whether the structures of well-conserved EP of plant ferritins are common in all plants, and (ii) whether the EP truly contributes to the shell stability of the plant ferritin oligomer. To clarify these matters, we have cloned a green-plant-type ferritin cDNA from a green alga, Ulva pertusa, and investigated its crystal structure. Ulva pertusa ferritin (UpFER) has a plant-ferritin-specific extension peptide composed of 28 amino acid residues. In the crystal structure of UpFER, the EP lay on and interacted with the neighboring threefold symmetry-related subunit. The amino acid residues involved in the interaction were very highly conserved among plant ferritins. The EPs masked the hydrophobic pockets on the ferritin shell surface by lying on them, and this made the ferritin oligomer more hydrophilic. Furthermore, differential scanning calorimetric analysis of the native and its EP-deletion mutant suggested that the EP contributed to the thermal stability of the plant ferritin shell. Thus, the shell stability and surface hydrophobicity of plant ferritin were controlled by the presence or absence of the plant-ferritin-specific EP. This regulation can account for those processes such as shell stability, degradation, and association of plant ferritin, which are significantly related to iron utilization in plants.     

Protein Association and Dissociation Regulated by Extension Peptide: A Mode for Iron Control by Phytoferritin in Seeds

Most of the iron in legume seeds is stored in ferritin located in the amyloplast, which is used during seed germination. However, there is a lack of information on the regulation of iron by phytoferritin. In this study, soluble and insoluble forms of pea (Pisum sativum) seed ferritin (PSF) isolated from dried seeds were found to be identical 24-mer ferritins comprising H-1 and H-2 subunits. The insoluble form is favored at low pH, whereas the two forms reversibly interconvert in the pH range of 6.0 to 7.8, with an apparent pK_a of 6.7. This phenomenon was not observed in animal ferritins, indicating that PSF is unique. The pH of the amyloplast was found to be approximately 6.0, thus facilitating PSF association, which is consistent with the role of PSF in long-term iron storage. Similar to previous studies, the results of this work showed that protein degradation occurs in purified PSF during storage, thus proving that phytoferritin also undergoes degradation during seedling germination. In contrast, no degradation was observed in animal ferritins, suggesting that this degradation of phytoferritin may be due to the extension peptide (EP), a specific domain found only in phytoferritin. Indeed, removal of EP from PSF significantly increased protein stability and prevented degradation under identical conditions while promoting protein dissociation. Correlated with such dissociation was a considerable increase in the rate of ascorbate-induced iron release from PSF at pH 6.0. Thus, phytoferritin may have facilitated the evolution of EP to enable it to regulate iron for storage or complement in seeds.Iron is an essential element for life, and its ability to form complexes, such as heme and iron-sulfur clusters in proteins and diiron enzymes, endows the cell with a variety of functions (Harrison and Arosio, 1996). However, free iron is toxic, because it facilitates the generation of highly reactive oxygen species that can damage cellular constituents. Therefore, iron homeostasis needs to be strictly controlled to avoid deficiency and toxicity, which are both known to dramatically impair the physiology of animals and plants, consequently affecting their development and growth.Ferritins are a class of intracellular iron storage and detoxification proteins that catalyze the oxidation of toxic ferrous ions by molecular oxygen or hydrogen peroxide to form a hydrous ferric oxide mineral core (Harrison and Arosio, 1996; Zhao, 2010). The importance of these proteins is reflected by their wide distribution throughout the animal, plant, and microbial kingdoms. Animal ferritins are generally heteropolymers composed of 24 subunits of two types, namely H and L, that assemble into a nearly spherical shell characterized by a 432-point symmetry. The diiron ferroxidase center located on the H-subunit of the mammalian protein is responsible for rapid Fe²⁺ oxidation, which consists of A and B iron-binding sites of conserved amino acid ligands His-65, Glu-27, Glu-107, and Glu-62. H-bonding residues, Gln-141 and Tyr-34, are nearby. In contrast, the L-subunit lacks such a ferroxidase center but contains a putative nucleation site consisting of a cluster of negative Glu-53, Glu-56, and Glu-57, which appears to be important for slower iron oxidation and mineralization (Crichton et al., 1996). The shape of either the H- or L-subunit is nearly cylindrical and composed of a four-α-helix bundle containing two antiparallel helix pairs (A, B and C, D) connected by a long nonhelical stretch, the BC loop (located between B- and C-helices). A fifth short helix (E-helix) lies at one end of the bundle, 60° to its axis (Harrison and Arosio, 1996).Although plant and animal ferritins arise from a common ancestor, plant ferritins exhibit various specific features as compared with animal analogs. Plant ferritins are seen in plastids, such as mitochondria in stems, chloroplasts in leaves, and amyloplasts in seeds, where iron is incorporated into the ferritin shell to form the mineral core (Laulhere et al., 1989; Waldo et al., 1995; Masuda et al., 2001; Zancani et al., 2004), whereas animal ferritins are located in the cellular cytoplasm (Harrison and Arosio, 1996). To date, all known phytoferritins contain only the H-type subunit but are controlled by multiple genes. Except for ferritin from Arabidopsis (Arabidopsis thaliana; Ravet et al., 2009), naturally occurring phytoferritins from soybean (Glycine max) seed (Masuda et al., 2001), pea (Pisum sativum) seed (Li et al., 2009a), alfalfa (Medicago sativa) seed (Barceló et al., 1997), and maize (Zea mays; Laulhere et al., 1988) consist of two different H-type subunits, which are designated H-1 and H-2 (26.5 and 28.0 kD, respectively), sharing approximately 80% amino acid sequence identity. The ratio of H-1 to H-2 in phytoferritin varies depending on the source (Laulhere et al., 1988; Barceló et al., 1997; Masuda et al., 2001; Li et al., 2009a). The two subunits are synthesized from a 32-kD precursor with a unique two-domain N-terminal sequence, the “transit peptide,” followed by the “extension peptide” (EP). The transit peptide is responsible for precursor targeting to plastids (Ragland et al., 1990), after which it is cleaved from the subunit precursor, leading to the formation of the mature subunit, which assembles into a 24-mer apoferritin within plastids (Ragland et al., 1990; Lescure et al., 1991). Thus, there are 24 EP domains per molecule of mature phytoferritin. In the case of pea seed ferritin (PSF), each EP domain is composed of 24 amino acid residues, 11 of which form a specific α-helix termed the P-helix and are flanked by Pro residues (X and L; Lobréaux et al., 1992).Iron oxidation at low iron flux into phytoferritin (48 or fewer iron atoms per protein shell) occurs exclusively in the diiron ferroxidase center. When the binding capacity of the diiron ferroxidase centers (48 iron atoms per protein shell) is exceeded, EP acts as a secondary binding and ferroxidase center that plays an important role in iron mineralization through reversible protein association/dissociation (Li et al., 2009a). The resultant iron core is stored within the ferritin shell. The amount of ferritin iron accounts for approximately 92% of total seed iron content (Marentesa and Grusaka, 1998), making it the major form of iron storage in seeds. This conclusion is supported by findings that ferritin subunits accumulate in legume seeds during maturation and remain in dry seeds (Lobréaux and Briat, 1991). Phytoferritin then appears to be in a totally different environment compared with animal ferritin. A previous study has shown that phytoferritin iron was used during seed germination and subsequent seedling growth (Lobréaux and Briat, 1991). However, information regarding the regulation of phytoferritin iron in seeds is still lacking up to the present. Since EP represents the major structural difference between animal and plant ferritins (Masuda et al., 2010), this study focuses on determining whether EP domains participate in the regulation of phytoferritin iron.This study investigates the effect of pH on ferritin association/dissociation. Transmission electron microscopy (TEM) will be used to directly observe associated PSF molecules. The stability of PSF and EP-deleted PSF during storage and protein degradation will also be evaluated in vitro. The results of this study may provide new insights into the regulation of phytoferritin iron in seeds through EP-influenced protein association/dissociation reactions.     

Could a dysfunction of ferritin be a determinant factor in the aetiology of some neurodegenerative diseases?

Background

The concentration of iron in the brain increases with aging. Furthermore, it has also been observed that patients suffering from neurological diseases (e.g. Parkinson, Alzheimer…) accumulate iron in the brain regions affected by the disease. Nevertheless, it is still not clear whether this accumulation is the initial cause or a secondary consequence of the disease. Free iron excess may be an oxidative stress source causing cell damage if it is not correctly stored in ferritin cores as a ferric iron oxide redox-inert form.

Scope

Both, the composition of ferritin cores and their location at subcellular level have been studied using analytical transmission electron microscopy in brain tissues from progressive supranuclear palsy (PSP) and Alzheimer disease (AD) patients.

Major conclusions

Ferritin has been mainly found in oligodendrocytes and in dystrophic myelinated axons from the neuropili in AD. In relation to the biomineralization of iron inside the ferritin shell, several different crystalline structures have been observed in the study of physiological and pathological ferritin. Two cubic mixed ferric–ferrous iron oxides are the major components of pathological ferritins whereas ferrihydrite, a hexagonal ferric iron oxide, is the major component of physiological ferritin. We hypothesize a dysfunction of ferritin in its ferroxidase activity.

General significance

The different mineralization of iron inside ferritin may be related to oxidative stress in olygodendrocites, which could affect myelination processes with the consequent perturbation of information transference.     

Releasing iron from ferritin protein nanocage by reductive method: The role of electron transfer mediator

Background

Ferritin detoxifies excess of free Fe(II) and concentrates it in the form of ferrihydrite (Fe₂O₃·xH₂O) mineral. When in need, ferritin iron is released for cellular metabolic activities. However, the low solubility of Fe(III) at neutral pH, its encapsulation by stable protein nanocage and presence of dissolved O₂ limits in vitro ferritin iron release.

Reactivity of ferritin and the structure of ferritin-derived ferrihydrite

Background

In nature or in the laboratory, the roughly spherical interior of the ferritin protein is well suited for the formation and storage of a variety of nanosized metal oxy-hydroxide compounds which hold promise for a range of applications. However, the linkages between ferritin reactivity and the structure and physicochemical properties of the nanoparticle core, either native or reconstituted, remain only partly understood.

Scope of review

Here we review studies, including those from our laboratory, which have investigated the structure of ferritin-derived ferrihydrite and reactivity of ferritin, both native and reconstituted. Selected proposed structure models for ferrihydrite are discussed along with the structural and genetic relationships that exist among several different forms of ferrihydrite. With regard to reactivity, the review will emphasize studies that have investigated the (photo)reactivity of ferritin and ferritin-derived materials with environmentally relevant gaseous and aqueous species.

Major conclusions

The inorganic core formed from apoferritin reconstituted with varied amounts of Fe has the same structural topology as the inorganically derived ferrihydrite that is an important component of many environmental and soil systems. Reactivity of ferritin toward aqueous species resulting from the photoexcitation of the inorganic core of the protein shows promise for driving redox reactions relevant to environmental chemistry.

General significance

Ferritin-derived ferrihydrite is effectively maintained in a relatively unaggregated state, which improves reactivity and opens the possibility of future applications in environmental remediation. Advances in our understanding of the structure, composition, and disorder in synthetic, inorganically derived ferrihydrite are shedding new light on the reactivity and stability of ferrihydrite derived artificially from ferritin.     

Phosphoinositide-3-kinase inhibition elevates ferritin level resulting depletion of labile iron pool and blocking of glioma cell proliferation

Background

Elevated endogenous phosphoinositide-3-kinase (PI3K) activity is critical for cell proliferation in gliomas. Iron availability is one of the essential factors for cell growth and proliferation. However, any relation between PI3K and cellular iron homeostasis has not been understood so far.

Iron induces ferritin synthesis in maize plantlets

The iron-storage protein ferritin has been purified to homogeneity from maize seeds, allowing to determine the sequence of the first 29 NH₂-terminal amino acids of its subunit and to raise specific rabbit polyclonal antibodies. Addition of 500 M Fe-EDTA/75 M Fe-citrate to hydroponic culture solutions of maize plantlets, previously starved for iron, led to a significant increase of the iron concentration of roots and leaves, albeit root iron was mainly found associated with the apoplast. Immunodetection of ferritin by western blots indicated that this iron treatment induced ferritin protein accumulation in roots and leaves over a period of 3 days. In order to investigate this induction at the ferritin mRNA level, various ferritin cDNA clones were isolated from a cDNA library prepared from poly(A)⁺ mRNA isolated from roots 48 h after iron treatment. These cDNAs were classified into two groups called FM1 and FM2. Upstream of the sequence encoding the mature ferritin subunit, both of these cDNAs contained an in-frame coding sequence with the characteristics of a transit peptide for plastid targeting. Two members of the FM1 subfamily, both partial at their 5 extremity, were characterized. They are identical, except in their 3 untranslated region: FM1A extends 162 nucleotides beyond the 3 terminus of FM1B. These two mRNAs could arise from the use of two different polyadenylation signals. FM2 is 96% identical to FM1 and contains 45 nucleotides of 5 untranslated region. Northern analyses of root and leaf RNAs, at different times after iron treatment, revealed ferritin mRNA accumulation in response to iron. Ferritin mRNA accumulation was transient and particularly abundant in leaves, reaching a maximum at 24 h. The level of ferritin mRNA in roots was affected to a lesser extent than in leaves.     

Detection of oxidized and glycated proteins in clinical samples using mass spectrometry — A user's perspective

Background

Proteins in human tissues and body fluids continually undergo spontaneous oxidation and glycation reactions forming low levels of oxidation and glycation adduct residues. Proteolysis of oxidised and glycated proteins releases oxidised and glycated amino acids which, if they cannot be repaired, are excreted in urine.

Scope of Review

In this review we give a brief background to the classification, formation and processing of oxidised and glycated proteins in the clinical setting. We then describe the application of stable isotopic dilution analysis liquid chromatography-tandem mass spectrometry (LC-MS/MS) for measurement of oxidative and glycation damage to proteins in clinical studies, sources of error in pre-analytic processing, corroboration with other techniques – including how this may be improved – and a systems approach to protein damage analysis for improved surety of analyte estimations.

Major conclusions

Stable isotopic dilution analysis LC-MS/MS provides a robust reference method for measurement of protein oxidation and glycation adducts. Optimised pre-analytic processing of samples and LC-MS/MS analysis procedures are required to achieve this.

General significance

Quantitative measurement of protein oxidation and glycation adducts provides information on level of exposure to potentially damaging protein modifications, protein inactivation in ageing and disease, metabolic control, protein turnover, renal function and other aspects of body function. Reliable and clinically assessable analysis is required for translation of measurement to clinical diagnostic use. Stable isotopic dilution analysis LC-MS/MS provides a “gold standard” approach and reference methodology to which other higher throughput methods such as immunoassay and indirect methods are preferably corroborated by researchers and those commercialising diagnostic kits and reagents. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.     

Methionine sulfoxide reductase A (MsrA) restores α-crystallin chaperone activity lost upon methionine oxidation

Background

Lens cataract is associated with protein oxidation and aggregation. Two proteins that cause cataract when deleted from the lens are methionine sulfoxide reductase A (MsrA) that repairs protein methionine sulfoxide (PMSO) oxidized proteins and α-crystallin which is a two-subunit (αA and αB) chaperone. Here, we tested whether PMSO formation damages α-crystallin chaperone function and whether MsrA could repair PMSO-α-crystallin.

Methods

Total α-crystallin was oxidized to PMSO and evaluated by CNBr-cleavage and mass spectrometry. Chaperone activity was measured by light scattering using lysozyme as target. PMSO-α-crystallin was treated with MsrA, and repair was assessed by CNBr cleavage, mass spectrometry and recovery of chaperone function. The levels of α-crystallin-PMSO in the lenses of MsrA-knockout relative to wild-type mice were determined.

Results

PMSO oxidation of total α-crystallin (met 138 of αA and met 68 of αB) resulted in loss of α-crystallin chaperone activity. MsrA treatment of PMSO-α-crystallin repaired its chaperone activity through reduction of PMSO. Deletion of MsrA in mice resulted in increased levels of PMSO-α-crystallin.

Conclusions

Methionine oxidation damages α-crystallin chaperone function and MsrA can repair PMSO-α-crystallin restoring its chaperone function. MsrA is required for maintaining the reduced state of α-crystallin methionines in the lens.

Significance

Methionine oxidation of α-crystallin in combination with loss of MsrA repair causes loss of α-crystallin chaperone function. Since increased PMSO levels and loss of α-crystallin function are hallmarks of cataract, these results provide insight into the mechanisms of cataract development and likely those of other age-related diseases.     

A plant peptide: N-glycanase orthologue facilitates glycoprotein ER-associated degradation in yeast

Background

The cytoplasmic peptide:N-glycanase (PNGase) is a deglycosylating enzyme involved in the ER-associated degradation (ERAD) process, while ERAD-independent activities are also reported. Previous biochemical analyses indicated that the cytoplasmic PNGase orthologue in Arabidopsis thaliana (AtPNG1) can function as not only PNGase but also transglutaminase, while its in vivo function remained unclarified.

Methods

AtPNG1 was expressed in Saccharomyces cerevisiae and its in vivo role on PNGase-dependent ERAD pathway was examined.

Results

AtPNG1 could facilitate the ERAD through its deglycosylation activity. Moreover, a catalytic mutant of AtPNG1 (AtPNG1(C251A)) was found to significantly impair the ERAD process. This result was found to be N-glycan-dependent, as the AtPNG(C251A) did not affect the stability of the non-glycosylated RTA? (ricin A chain non-toxic mutant). Tight interaction between AtPNG1(C251A) and the RTA? was confirmed by co-immunoprecipitation analysis.

Conclusion

The plant PNGase facilitates ERAD through its deglycosylation activity, while the catalytic mutant of AtPNG1 impair glycoprotein ERAD by binding to N-glycans on the ERAD substrates.

General significance

Our studies underscore the functional importance of a plant PNGase orthologue as a deglycosylating enzyme involved in the ERAD.     

Selective cytotoxicity of intense nanosecond-duration electric pulses in mammalian cells

Background

Nanosecond electric pulses (EP) disrupt cell membrane and organelles and cause cell death in a manner different from the conventional irreversible electroporation. We explored the cytotoxic effect of 10-ns EP (quantitation, mechanisms, efficiency, and specificity) in comparison with 300-ns, 1.8- and 9-μs EP.

Methods

Effects in Jurkat and U937 cells were characterized by survival assays, DNA electrophoresis and flow cytometry.

Results

10-ns EP caused apoptotic or necrotic death within 2–20 h. Survival (S, %) followed the absorbed dose (D, J/g) as: S = αD^(−K), where coefficients K and α determined the slope and the “shoulder” of the survival curve. K was similar in all groups, whereas α was cell type- and pulse duration-dependent. Long pulses caused immediate propidium uptake and phosphatidylserine (PS) externalization, whereas 10-ns pulses caused PS externalization only.

Conclusions

1.8- and 9-μs EP cause cell death efficiently and indiscriminately (LD₅₀ 1–3 J/g in both cell lines); 10-ns EP are less efficient, but very selective (LD₅₀ 50–80 J/g for Jurkat and 400–500 J/g for U937); 300-ns EP show intermediate effects. Shorter EP open propidium-impermeable, small membrane pores (”nanopores”), triggering different cell death mechanisms.

General significance

Nanosecond EP can selectively target certain cells in medical applications like tumor ablation.