Complex Nature of Protein Carbonylation Specificity After Metal-Catalyzed Oxidation.

PURPOSE: Protein carbonylation is an irreversible modification of Lys, Arg, Thr and Pro amino acids under conditions of oxidative stress. Previous studies have reported specific carbonylated residues in purified recombinant albumins, albeit with a lack of agreement between the studies. Currently, structural factors that determine site-specific protein carbonylation are not well understood. METHODS: In this study, we utilized metal-catalyzed oxidizing conditions to generate carbonylation in recombinant human serum albumin (HSA) and granulocyte-colony stimulating factor (G-CSF), two proteins with distinct metal-binding abilities. To estimate predictability of HSA carbonylation sites, the same oxidative reaction was repeated based on the previously reported conditions. For G-CSF, oxidative conditions were gradually adjusted to achieve substantial levels of protein carbonylation. Corresponding accumulation of specific oxidized residues was identified and confirmed with high-resolution mass spectrometry. RESULTS: Our HSA dataset contained 55 carbonylated residues and showed a significant overlap with the previously published pooled data, indicating a certain level of carbonylation site specificity for albumins. Oxidation of G-CSF under multiple oxidative conditions consistently showed a highly specific carbonylation at position Pro45. We also detected a previously unreported, oxidation-induced cleavage site in G-CSF between His44 and Pro45, which might be attributed to a presence of a potential metal-binding site near residue Pro45. CONCLUSIONS: Our results show distinct patterns of protein carbonylation for HSA and G-CSF. Thus, specificity of protein carbonylation induced by metal-catalyzed oxidation is protein dependent and might be predicted by availability of transition metal binding site(s) within the protein.

Hepatic expression of serum amyloid A1 is induced by traumatic brain injury and modulated by telmisartan.

Traumatic brain injury affects the whole body in addition to the direct impact on the brain. The systemic response to trauma is associated with the hepatic acute-phase response. To further characterize this response, we performed controlled cortical impact injury on male mice and determined the expression of serum amyloid A1 (SAA1), an apolipoprotein, induced at the early stages of the acute-phase response in liver and plasma. After cortical impact injury, induction of SAA1 was detectable in plasma at 6 hours post-injury and in liver at 1 day post-injury, followed by gradual diminution over time. In the liver, cortical impact injury increased neutrophil and macrophage infiltration, apoptosis, and expression of mRNA encoding the chemokines CXCL1 and CXCL10. An increase in angiotensin II AT1 receptor mRNA at 3 days post-injury was also observed. Administration of the AT1 receptor antagonist telmisartan 1 hour post-injury significantly decreased liver SAA1 levels and CXCL10 mRNA expression, but did not affect CXCL1 expression or the number of apoptotic cells or infiltrating leukocytes. To our knowledge, this is the first study to demonstrate that SAA1 is induced in the liver after traumatic brain injury and that telmisartan prevents this response. Elucidating the molecular pathogenesis of the liver after brain injury will assist in understanding the efficacy of therapeutic approaches to brain injury.

Comparative Effects of Metal-Catalyzed Oxidizing Systems on Carbonylation and Integrity of Therapeutic Proteins.

PURPOSE: Ascorbic acid has been considered as a potential radical scavenging excipient for pharmaceutical formulations. However, under certain circumstances, ascorbic acid can generate reactive oxygen species via redox cycling. The objective of this study was to investigate ascorbic acid-induced oxidative carbonylation of therapeutic proteins and correlate the increase in carbonylation with protein aggregation. METHODS: An optimized ELISA for quantifying carbonyl levels was used to compare the oxidizing potentials of ascorbic acid and hydrogen peroxide by testing four pharmaceutically-relevant proteins (human serum albumin, immunoglobulin G, granulocyte-colony stimulating factor and calcitonin). Several transition metals at micromolar concentrations were evaluated for their ability to enhance ascorbic acid-induced protein carbonylation. Protein aggregation under oxidative conditions, with or without free radical scavengers, was measured by aggregate binding fluorescent dye and confirmed by microfluidic imaging. RESULTS: Addition of ascorbic acid alone resulted in higher increases in carbonylation than addition of hydrogen peroxide. The presence of trace amounts (>75 ppb) of copper enhanced oxidative effects of ascorbic acid, whereas other tested metals did not comparably promote oxidation. During oxidation, protein destabilization indicated by loss of the full-length protein, positively correlated with the increase in protein aggregation. However, levels of aggregation did not always correlate with the levels of protein carbonylation. At comparable carbonylation levels, addition of copper produced greater protein destabilization and aggregation than addition of iron. CONCLUSIONS: The results strongly suggest that ascorbic acid with traces of metals, especially copper, can promote therapeutic protein carbonylation and potentially aggregation. At similar carbonylation levels, some oxidative conditions may lead to greater protein destabilization than others.

Sex, prions, and plasmids in yeast.

Even deadly prions may be widespread in nature if they spread by infection faster than they kill off their hosts. The yeast prions [PSI+] and [URE3] (amyloids of Sup35p and Ure2p) were not found in 70 wild strains, while [PIN+] (amyloid of Rnq1p) was found in ∼16% of the same population. Yeast prion infection occurs only by mating, balancing the detrimental effects of carrying the prion. We estimated the frequency of outcross mating as about 1% of mitotic doublings from the known detriment of carrying the 2-µm DNA plasmid (∼1%) and its frequency in wild populations (38/70). We also estimated the fraction of total matings that are outcross matings (∼23-46%) from the fraction of heterozygosity at the highly polymorphic RNQ1 locus (∼46%). These results show that the detriment of carrying even the mildest forms of [PSI+], [URE3], or [PIN+] is greater than 1%. We find that Rnq1p polymorphisms in wild strains include several premature stop codon alleles that cannot propagate [PIN+] from the reference allele and others with several small deletions and point mutations which show a small transmission barrier. Wild strains carrying [PIN+] are far more likely to be heterozygous at RNQ1 and other loci than are [pin-] strains, probably reflecting its being a sexually transmitted disease. Because sequence differences are known to block prion propagation or ameliorate its pathogenic effects, we hypothesize that polymorphism of RNQ1 was selected to protect cells from detrimental effects of the [PIN+] prion.

Non-targeted identification of prions and amyloid-forming proteins from yeast and mammalian cells.

The formation of amyloid aggregates is implicated both as a primary cause of cellular degeneration in multiple human diseases and as a functional mechanism for providing extraordinary strength to large protein assemblies. The recent identification and characterization of several amyloid proteins from diverse organisms argues that the amyloid phenomenon is widespread in nature. Yet identifying new amyloid-forming proteins usually requires a priori knowledge of specific candidates. Amyloid fibers can resist heat, pressure, proteolysis, and denaturation by reagents such as urea or sodium dodecyl sulfate. Here we show that these properties can be exploited to identify naturally occurring amyloid-forming proteins directly from cell lysates. This proteomic-based approach utilizes a novel purification of amyloid aggregates followed by identification by mass spectrometry without the requirement for special genetic tools. We have validated this technique by blind identification of three amyloid-based yeast prions from laboratory and wild strains and disease-related polyglutamine proteins expressed in both yeast and mammalian cells. Furthermore, we found that polyglutamine aggregates specifically recruit some stress granule components, revealing a possible mechanism of toxicity. Therefore, core amyloid-forming proteins as well as strongly associated proteins can be identified directly from cells of diverse origin.

Suicidal [PSI+] is a lethal yeast prion.

[PSI(+)] is a prion of the essential translation termination factor Sup35p. Although mammalian prion infections are uniformly fatal, commonly studied [PSI(+)] variants do not impair growth, leading to suggestions that [PSI(+)] may protect against stress conditions. We report here that over half of [PSI(+)] variants are sick or lethal. These "killer [PSI(+)]s" are compatible with cell growth only when also expressing minimal Sup35C, lacking the N-terminal prion domain. The severe detriment of killer [PSI(+)] results in rapid selection of nonkiller [PSI(+)] variants or loss of the prion. We also report variants of [URE3], a prion of the nitrogen regulation protein Ure2p, that grow much slower than ure2Δ cells. Our findings give a more realistic picture of the impact of the prion change than does focus on "mild" prion variants.

Prion diseases of yeast: amyloid structure and biology.

Prion "variants" or "strains" are prions with the identical protein sequence, but different characteristics of the prion infection: e.g. different incubation periods for scrapie strains or different phenotype intensities for yeast prion variants. We have shown that infectious amyloids of the yeast prions [PSI+], [URE3] and [PIN+] each have an in-register parallel ß-sheet architecture. Moreover, we have pointed out that this amyloid architecture can explain how one protein can faithfully transmit any of several conformations to new protein monomers. This explains how proteins can be genes.

A yeast model of optineurin proteinopathy reveals a unique aggregation pattern associated with cellular toxicity.

Many neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) are linked to the accumulation of specific protein aggregates in affected regions of the nervous system. SOD1, TDP-43, FUS and optineurin (OPTN) proteins were identified to form intraneuronal inclusions in ALS patients. In addition, mutations in OPTN are associated with both ALS and glaucoma. As the pathological role of OPTN in neuronal degeneration remains unresolved, we created a yeast model to study its potential for aggregation and toxicity. We observed that both wild type and disease-associated mutants of OPTN form toxic non-amyloid aggregates in yeast. Similar to reported cell culture and mouse models, the OPTN E50K mutant shows enhanced toxicity in yeast, implying a conserved gain-of-function mechanism. Furthermore, OPTN shows a unique aggregation pattern compared to other disease-related proteins in yeast. OPTN aggregates colocalize only partially with the insoluble protein deposit (IPOD) site markers, but coincide perfectly with the prion seed-reducing protein Btn2 and several other aggregation-prone proteins, suggesting that protein aggregates are not limited to a single IPOD site. Importantly, changes in the Btn2p level modify OPTN toxicity and aggregation. This study generates a mechanistic framework for investigating how OPTN may trigger pathological changes in ALS and other OPTN-linked neurodegenerative disorders.

Curing of the [URE3] prion by Btn2p, a Batten disease-related protein.

[URE3] is a prion (infectious protein), a self-propagating amyloid form of Ure2p, a regulator of yeast nitrogen catabolism. We find that overproduction of Btn2p, or its homologue Ypr158 (Cur1p), cures [URE3]. Btn2p is reported to be associated with late endosomes and to affect sorting of several proteins. We find that double deletion of BTN2 and CUR1 stabilizes [URE3] against curing by several agents, produces a remarkable increase in the proportion of strong [URE3] variants arising de novo and an increase in the number of [URE3] prion seeds. Thus, normal levels of Btn2p and Cur1p affect prion generation and propagation. Btn2p-green fluorescent protein (GFP) fusion proteins appear as a single dot located close to the nucleus and the vacuole. During the curing process, those cells having both Ure2p-GFP aggregates and Btn2p-RFP dots display striking colocalization. Btn2p curing requires cell division, and our results suggest that Btn2p is part of a system, reminiscent of the mammalian aggresome, that collects aggregates preventing their efficient distribution to progeny cells.

The yeast Sup35NM domain propagates as a prion in mammalian cells.

Prions are infectious, self-propagating amyloid-like protein aggregates of mammals and fungi. We have studied aggregation propensities of a yeast prion domain in cell culture to gain insights into general mechanisms of prion replication in mammalian cells. Here, we report the artificial transmission of a yeast prion across a phylogenetic kingdom. HA epitope-tagged yeast Sup35p prion domain NM was stably expressed in murine neuroblastoma cells. Although cytosolically expressed NM-HA remained soluble, addition of fibrils of bacterially produced Sup35NM to the medium efficiently induced appearance of phenotypically and biochemically distinct NM-HA aggregates that were inherited by daughter cells. Importantly, NM-HA aggregates also were infectious to recipient mammalian cells expressing soluble NM-HA and, to a lesser extent, to yeast. The fact that the yeast Sup35NM domain can propagate as a prion in neuroblastoma cells strongly argues that cellular mechanisms support prion-like inheritance in the mammalian cytosol.

Prion amyloid structure explains templating: how proteins can be genes.

The yeast and fungal prions determine heritable and infectious traits, and are thus genes composed of protein. Most prions are inactive forms of a normal protein as it forms a self-propagating filamentous ß-sheet-rich polymer structure called amyloid. Remarkably, a single prion protein sequence can form two or more faithfully inherited prion variants, in effect alleles of these genes. What protein structure explains this protein-based inheritance? Using solid-state nuclear magnetic resonance, we showed that the infectious amyloids of the prion domains of Ure2p, Sup35p and Rnq1p have an in-register parallel architecture. This structure explains how the amyloid filament ends can template the structure of a new protein as it joins the filament. The yeast prions [PSI(+)] and [URE3] are not found in wild strains, indicating that they are a disadvantage to the cell. Moreover, the prion domains of Ure2p and Sup35p have functions unrelated to prion formation, indicating that these domains are not present for the purpose of forming prions. Indeed, prion-forming ability is not conserved, even within Saccharomyces cerevisiae, suggesting that the rare formation of prions is a disease. The prion domain sequences generally vary more rapidly in evolution than does the remainder of the molecule, producing a barrier to prion transmission, perhaps selected in evolution by this protection.

Protein inheritance (prions) based on parallel in-register beta-sheet amyloid structures.

Most prions (infectious proteins) are self-propagating amyloids (filamentous protein multimers), and have been found in both mammals and fungal species. The prions [URE3] and [PSI+] of yeast are disease agents of Saccharomyces cerevisiae while [Het-s] of Podospora anserina may serve a normal cellular function. The parallel in-register beta-sheet structure shown by prion amyloids makes possible a templating action at the end of filaments which explains the faithful transmission of variant differences in these molecules. This property of self-reproduction, in turn, allows these proteins to act as de facto genes, encoding heritable information.

Nucleotide exchange factors for Hsp70s are required for [URE3] prion propagation in Saccharomyces cerevisiae.

The [URE3] and [PSI(+)] prions are infectious amyloid forms of Ure2p and Sup35p. Several chaperones influence prion propagation: Hsp104p overproduction destabilizes [PSI(+)], whereas [URE3] is sensitive to excess of Ssa1p or Ydj1p. Here, we show that overproduction of the chaperone, Sse1p, can efficiently cure [URE3]. Sse1p and Fes1p are nucleotide exchange factors for Ssa1p. Interestingly, deletion of either SSE1 or FES1 completely blocked [URE3] propagation. In addition, deletion of SSE1 also interfered with [PSI(+)] propagation.

Acute total body ionizing gamma radiation induces long-term adverse effects and immediate changes in cardiac protein oxidative carbonylation in the rat.

Radiation-induced heart disease presents a significant challenge in the event of an accidental radiation exposure as well as to cancer patients who receive acute doses of irradiation as part of radiation therapy. We utilized the spontaneously hypertensive Wistar-Kyoto rat model, previously shown to demonstrate drug-induced cardiomyopathy, to evaluate the acute and long-term effects of sub-lethal total body gamma irradiation at two, four, and fifty-two weeks. We further examined irreversible oxidative protein carbonylation in the heart immediately following irradiation in the normotensive Wistar-Kyoto rat. Both males and females sustained weight loss and anemic conditions compared to untreated controls over a one-year period as reflected by reduced body weight and low red blood cell count. Increased inflammation was detected by elevated IL-6 serum levels selectively in males at four weeks. Serum cardiac troponin T and I analyses revealed signs of cardiomyopathy at earlier timepoints, but high variability was observed, especially at one year. Echocardiography at two weeks following 5.0Gy treatment revealed a significant decrease in cardiac output in females and a significant decrease in both diastolic and systolic volumes in males. Following 10.0Gy irradiation in the normotensive Wistar-Kyoto rat, the heart tissue showed an increase in total protein oxidative carbonylation accompanied by DNA damage indicated by an increase in γ-H2AX. Using proteomic analyses, we identified several novel proteins which showed a marked difference in carbonylation including those of mitochondrial origin and most notably, cardiac troponin T, one of the key proteins involved in cardiomyocyte contractility. Overall, we present findings of acute oxidative protein damage, DNA damage, cardiac troponin T carbonylation, and long-term cardiomyopathy in the irradiated animals.

Two prion variants of Sup35p have in-register parallel beta-sheet structures, independent of hydration.

The [PSI(+)] prion is a self-propagating amyloid of the Sup35 protein, normally a subunit of the translation termination factor, but impaired in this vital function when in the amyloid form. The Sup35 N, M, and C domains are the amino-terminal prion domain, a connecting polar domain, and the essential C-terminal domain resembling eukaryotic elongation factor 1alpha respectively. Different [PSI(+)] isolates (prion variants) may have distinct biological properties, associated with different amyloid structures. Here we use solid state NMR to examine the structure of infectious Sup35NM amyloid fibrils of two prion variants. We find that both variants have an in-register parallel beta-sheet structure, both in the fully hydrated form and in the lyophilized form. Moreover, we confirm that some leucine residues in the M domain participate in the in-register parallel beta-sheet structure. Transmission of the [PSI(+)] prion by amyloid fibrils of Sup35NM and transmission of the [URE3] prion by amyloid fibrils of recombinant full-length Ure2p are similar whether they have been lyophilized or not (wet or dry).

Study of Amyloids Using Yeast.

We detail some of the genetic, biochemical, and physical methods useful in studying amyloids in yeast, particularly the yeast prions. These methods include cytoduction (cytoplasmic mixing), infection of cells with prion amyloids, use of green fluorescent protein fusions with amyloid-forming proteins for cytology, protein purification and amyloid formation, and electron microscopy of filaments.

Isolation and Analysis of Prion and Amyloid Aggregates from Yeast Cells.

Amyloid fibers are large and extremely stable structures that can resist denaturation by strong anionic detergents, such as sodium dodecyl sulfate or sarkosyl. Here, we present two complementary analytical methods that exploit these properties, enabling the isolation and characterization of amyloid/prion aggregates. The first technique, known as semidenaturating detergent agarose gel electrophoresis, is an immunoblotting technique, conceptually similar to conventional western blotting. It enables the targeted identification of large detergent-resistant protein aggregates using antibodies specific to the protein of interest. The second method, called the technique for amyloid purification and identification, is a nontargeted approach that can isolate amyloid aggregates for analysis by tandem mass spectrometry. The latter approach requires no special genetic tools or antibodies, and can identify amyloid-forming proteins, such as prions, as well as proteins tightly associated with amyloid, from a variety of cell sources.

Genetic Methods for Studying Yeast Prions.

The recognition that certain long-known nonchromosomal genetic elements were actually prions was based not on the specific phenotypic manifestations of those elements, but rather on their unusual genetic properties. Here, we outline methods of prion assay, methods for showing the nonchromosomal inheritance, and methods for determining whether a nonchromosomal trait has the unusual characteristics diagnostic of a prion. Finally, we discuss genetic methods often useful in the study of yeast prions.

Prion Transfection of Yeast.

Transfection of yeast with amyloid filaments, made from recombinant protein or prepared from extracts of cells infected with a prion, has become an important method in characterizing yeast prions. Here, we describe a method for transmission of [URE3] with Ure2p amyloid that is based on a previously published protocol for transfection with Sup35p filaments to make cells [PSI+]. This method may be used for other prions by changing just the amyloid source, host strain, and plating medium.

Prions.

Infectious proteins (prions) are usually self-templating filamentous protein polymers (amyloids). Yeast prions are genes composed of protein and, like the multiple alleles of DNA-based genes, can have an array of "variants," each a distinct self-propagating amyloid conformation. Like the lethal mammalian prions and amyloid diseases, yeast prions may be lethal, or only mildly detrimental, and show an array of phenotypes depending on the protein involved and the prion variant. Yeast prions are models for both rare mammalian prion diseases and for several very common amyloidoses such as Alzheimer's disease, type 2 diabetes, and Parkinson's disease. Here, we describe their detection and characterization using genetic, cell biological, biochemical, and physical methods.