Cyclotides Chemosensitize Glioblastoma Cells to Temozolomide.

Glioblastoma multiforme (GBM) is the most aggressive cancer originating in the brain, with a median survival of 12 months. Most patients do not respond to or develop resistance to the only effective chemotherapeutic drug, temozolomide (TMZ), used to treat gliomas. Novel treatment methods are critically needed. Cyclotides are plant peptides that may be promising adjuvants to TMZ chemotherapy. They exhibit antitumor activity and chemosensitize cells to doxorubicin in breast cancer studies. During this research, we optimized cyclotide isolation techniques, and several cyclotides (CyO2, CyO13, kalata B1, and varv peptide A) exhibited dose-dependent cytotoxicity in MTT assays with IC50 values of 2.15-7.92 µM against human brain astrocytoma cells (U-87 MG) and human bone marrow derived neuroblastoma cells (SH-SY5Y). CyO2 and varv peptide A increased TMZ-induced cell death in U-87 MG cultures alone and when coexposed with CyO2 or varv peptide A plus TMZ. Phase contrast microscopy of glioblastoma cells exposed to cyclotides alone and coexposed to TMZ indicated shrunken, granular cells with blebbing, and the most pronounced effects were observed with coexposure treatments of cyclotides and TMZ. Cumulative results provide the proof-of-concept that cyclotides may enhance TMZ chemotherapy, and in vivo pharmacokinetic investigations of cyclotides are warranted with respect to GBM.

Mechanisms of L-Serine-Mediated Neuroprotection Include Selective Activation of Lysosomal Cathepsins B and L.

L-serine is a naturally occurring dietary amino acid that has recently received renewed attention as a potential therapy for the treatment of amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), hereditary sensory autonomic neuropathy type I (HSAN1), and sleep induction and maintenance. We have previously reported L-serine functions as a competitive inhibitor of L-BMAA toxicity in cell cultures and have since progressed to examine the neuroprotective effects of L-serine independent of L-BMAA-induced neurotoxicity. For example, in a Phase I, FDA-approved human clinical trial of 20 ALS patients, our lab reported 30 g L-serine/day was safe, well-tolerated, and slowed the progression of the disease in a group of 5 patients. Despite increasing evidence for L-serine being useful in the clinic, little is known about the mechanism of action of the observed neuroprotection. We have previously reported, in SH-SY5Y cell cultures, that L-serine alone can dysregulate the unfolded protein response (UPR) and increase the translation of the chaperone protein disulfide isomerase (PDI), and these mechanisms may contribute to the clearance of mis- or unfolded proteins. Here, we further explore the pathways involved in protein clearance when L-serine is present in low and high concentrations in cell culture. We incubated SH-SY5Y cells in the presence and absence of L-serine and measured changes in the activity of proteolytic enzymes from the autophagic-lysosomal system, cathepsin B, cathepsin L, and arylsulfatase and specific activities of the proteasome, peptidylglutamyl-peptide hydrolyzing (PGPH) (also called caspase-like), chymotrypsin, and trypsin-like. Under our conditions, we report that L-serine selectively induced the activity of autophagic-lysosomal enzymes, cathepsins B and L, but not any of the proteasome-hydrolyzing activities. To enable comparison with previous work, we also incubated cells with L-BMAA and report no effect on the activity of the autophagic lysosomes or the proteasomes. We also developed an open-source script for the automation of linear regression calculations of kinetic data. Autophagy impairment or failure is characteristic of many neurodegenerative disease; thus, activation of autophagic-lysosomal proteolysis may contribute to the neuroprotective effect of L-serine, which has been reported in cell culture and human clinical trials.

The Cyanotoxin and Non-protein Amino Acid ß-Methylamino-L-Alanine (L-BMAA) in the Food Chain: Incorporation into Proteins and Its Impact on Human Health.

The size and frequency of cyanobacterial blooms are increasing concomitantly with rising global temperatures and increased eutrophication, and this has implications for human health. Cyanotoxins, including L-BMAA, have been implicated in triggering neurodegenerative diseases such as ALS/PDC and Alzheimer's disease. L-BMAA is a water-soluble non-protein amino acid that can bioaccumulate up the food chain, in a free- and protein-bound form. While some data exists on the degree of environmental enrichment of L-BMAA in water bodies, cyanobacteria-derived supplements, fruit bats, and seafood, virtually nothing is known about the presence of L-BMAA in other foodstuffs. It has now been shown several times in laboratory settings that plants can absorb L-BMAA into their leaves and stems, but data from wild-grown plants is nascent. One of the mechanisms implicated in L-BMAA bioaccumulation is misincorporation into proteins in the place of the canonical amino acid L-serine. We first identified this as a mechanism of action of L-BMAA in 2013, and since then, several groups have replicated these findings, but others have not. Here, we discuss in detail the experimental approaches, why they may have produced negative findings and propose several ways forward for developing consistency within the field. We emphasize the need to standardize cell culture methods, using L-serine-free medium to study misincorporation of BMAA, and urge accurate reporting of the components present in cell culture media.

The use of L-serine to prevent ß-methylamino-L-alanine (BMAA)-induced proteotoxic stress in vitro.

ß-methylamino-L-alanine (BMAA), a non-protein amino acid synthesised by cyanobacteria, has been linked to a complex neurological disorder on Guam and more recently to other cases of sporadic ALS (sALS), however the mechanisms of BMAA toxicity are not completely understood. We have previously demonstrated that BMAA is misincorporated into newly synthesised proteins by human neuroblastoma cells and fibroblasts, resulting in the formation of autofluorescent material and the induction of apoptotic cell death. In the present study we show that BMAA at low levels does not cause an acute toxicity in neuroblastoma cells but increases the expression of the ER stress marker, C/EBP homologous protein (CHOP) and increases the activity of the pro-apoptotic enzyme caspase-3. We also observed an increase in the activity of the lysosomal cysteine proteases cathepsin B and L, characteristic of the accumulation of proteins in the lysosomal system. We were able to prevent these proteotoxic effects in neuroblastoma cells through co-treatment with l-serine suggesting that they resulted from incorporation of BMAA into proteins. Misincorporation provides a possible mechanism whereby BMAA could initiate misfolding, and the accumulation of aggregate-prone proteins in neurons. This build-up of misfolded proteins could explain the long latency period of the disease previously reported on Guam.

Using an in vitro model to study oxidised protein accumulation in ageing fibroblasts.

BACKGROUND: The accumulation of oxidised proteins in ageing cells and tissues results from an increase in oxidant damage coupled with impaired degradation of the damaged proteins. Heat Shock Proteins (HSP) and other chaperones are required to recognise damaged proteins and transport them to the lysosomal and proteasomal degradation pathways. How these systems fail in ageing cells is not clear. METHODS: We monitor oxidised protein accumulation, the activity of the proteasome and lysosomal proteases, and HSP levels in MRC-5 fibroblasts throughout their mitotic lifespan. We then use a novel in vitro cell culture model to experimentally generate oxidised proteins in young and old MRC-5 fibroblasts and compare their rates of degradation and changes in the key pathways involved in oxidised protein removal. RESULTS: We show that the activity of the proteasome and some lysosomal enzymes decreases with ageing in MRC-5 cells as do levels of HSP70 but this is not associated with an accumulation of oxidised proteins which only occurs as cells closely approach post-mitotic senescence. Old cells are unable to degrade experimentally generated oxidised proteins as efficiently as young cells. Exposure to mild heat stress however increases the efficiency of oxidised protein degradation by young cells and increases levels of HSP70. CONCLUSIONS: Our results highlight the importance of the HSP/chaperone system in oxidised protein metabolism, particularly in ageing cells. GENERAL SIGNIFICANCE: These data might have implications for the development of therapies for pathologies associated with protein accumulation and suggest that the HSP/chaperone system would be an important target.

Revisiting adverse reactions to vaccines: A critical appraisal of Autoimmune Syndrome Induced by Adjuvants (ASIA).

In 2011 Shoenfeld and Agmon-Levin proposed a new syndrome as a way of grouping together a range of emerging autoimmune diseases with possible adjuvant-associated causes, Autoimmune/Auto-inflammatory Syndrome Induced by Adjuvants (ASIA). At present, there is no evidence to suggest that ASIA syndrome is a viable explanation for unusual autoimmune diseases. Since the initial paper, over 80 publications have discussed ASIA. This systematic review examines the research that has been done to investigate whether ASIA is a broad umbrella term with little clinical significance, or whether there is some underlying mechanism which could be utilised to reduce the occurrence of adjuvant mediated disease. Twenty-seven animal, epidemiological and case studies were reviewed. Unfortunately, a robust animal model of ASIA using biologically relevant doses of adjuvants has yet to be defined. It is also apparent that the broadness of the current ASIA criteria lack stringency and, as a result, very few cases of autoimmune disease could be excluded from a diagnosis of ASIA. The current studies involving human cases are so diverse, in both external stimuli and in resulting conditions, that there is currently a lack of reproducible evidence for any consistent relationship between adjuvant and autoimmune condition. The addition of a mandatory criterion requiring temporal association and clinically relevant adjuvant dose would allow better definition of what constitutes a diagnosis of ASIA.

L-DOPA is incorporated into brain proteins of patients treated for Parkinson's disease, inducing toxicity in human neuroblastoma cells in vitro.

Levodopa (L-DOPA), a close structural analogue of the protein amino acid L-tyrosine, can substitute for L-tyrosine in protein synthesis and be mistakenly incorporated into newly synthesised proteins in vitro. We show that L-DOPA-containing proteins are present in the brain in L-DOPA-treated Parkinson's disease patients and accumulate in specific brain regions. In vitro studies demonstrate that substitution of L-tyrosine residues in proteins with L-DOPA causes protein misfolding and promotes protein aggregation in SH-SY5Y neuroblastoma cells resulting in the appearance of autofluorescent bodies. We show that the presence of L-DOPA-containing proteins causes profound changes in mitochondria and stimulates the formation of autophagic vacuoles in cells. Unlike L-DOPA, which is toxic to cells through its ability to generate radicals, proteins containing incorporated L-DOPA are toxic to SH-SY5Y cells by a mechanism independent of oxidative stress and resistant to antioxidants. These data suggest that the accumulation of L-DOPA-containing proteins in vulnerable cells might negatively impact on cell function.

Proteins containing oxidized amino acids induce apoptosis in human monocytes.

Cellular deposits of oxidized and aggregated proteins are hallmarks of a variety of age-related disorders, but whether such proteins contribute to pathology is not well understood. We previously reported that oxidized proteins form lipofuscin/ceroid-like bodies with a lysosomal-type distribution and up-regulate the transcription and translation of proteolytic lysosomal enzymes in cultured J774 mouse macrophages. Given the recently identified role of lysosomes in the induction of apoptosis, we have extended our studies to explore a role for oxidized proteins in apoptosis. Oxidized proteins were biosynthetically generated in situ by substituting oxidized analogues for parent amino acids. Apoptosis was measured with Annexin-V/PI (propidium iodide), TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling), MMP (mitochondrial membrane permeabilization), caspase activation and cytochrome c release, and related to lysosomal membrane permeabilization. Synthesized proteins containing the tyrosine oxidation product L-DOPA (L-3,4-dihydroxyphenylalanine) were more potent inducers of apoptosis than proteins containing the phenylalanine oxidation product o-tyrosine. Apoptosis was dependent upon incorporation of oxidized residues, as indicated by complete abrogation in cultures incubated with the non-incorporation control D-DOPA (D-3,4-dihydroxyphenylalanine) or when incorporation was competed out by parent amino acids. The findings of the present study suggest that certain oxidized proteins could play an active role in the progression of age-related disorders by contributing to LMP (lysosomal membrane permeabilization)-initiated apoptosis and may have important implications for the long-term use of L-DOPA as a therapeutic agent in Parkinson's disease.

Oxidized proteins: mechanisms of removal and consequences of accumulation.

Elevated levels of oxidized proteins are reported in diseased tissue from age-related pathologies such as atherosclerosis, neurodegenerative disorders, and cataract. Unlike the precise mechanisms that exist for the repair of nucleic acids, lipids, and carbohydrates, the primary pathway for the repair of oxidized proteins is complete catabolism to their constitutive amino acids. This process can be inefficient as is evidenced by their accumulation. It is generally considered that damaged proteins are degraded by the proteasome; however, this is only true for mildly oxidized proteins, because substrates must be unfolded to enter the narrow catalytic core. Rather, evidence suggests that moderately or heavily oxidized proteins are endocytosed and enter the endosomal/lysosomal system, indicating co-operation between the proteasomes and the lysosomes. Heavily modified substrates are incompletely degraded and accumulate within the lysosomal compartments resulting in the formation of lipofuscin-like, autofluorescent aggregates. Accumulation eventually results in impaired turnover of large organelles such as proteasomes and mitochondria, lysosomal destablization, leakage of proteases into the cytosol and apoptosis. In this review, we summarize reports published since our last assessments of the field of oxidized protein degradation including a role for modified proteins in the induction of apoptosis.

The impact of specific oxidized amino acids on protein turnover in J774 cells.

Oxidized protein deposition and accumulation have been implicated in the aetiology of a wide variety of age-related pathologies. Protein oxidation in vivo commonly results in the in situ modification of amino acid side chains, generating new oxidized amino acid residues in proteins. We have demonstrated previously that certain oxidized amino acids can be (mis)incorporated into cell proteins in vitro via protein synthesis. In the present study, we show that incorporation of o- and m-tyrosine resulted in increased protein catabolism, whereas dopa incorporation generated proteins that were inefficiently degraded by cells. Incorporation of higher levels of L-dopa into proteins resulted in an increase in the activity of lysosomal cathepsins, increased autofluorescence and the generation of high-molecular-mass SDS-stable complexes, indicative of protein aggregation. These effects were due to proteins containing incorporated L-dopa, since they were not seen with the stereoisomer D-dopa, which enters the cell and generates the same reactive species as L-dopa, but cannot be incorporated into proteins. The present study highlights how the nature of the oxidative modification to the protein can determine the efficiency of its removal from the cell by proteolysis. Protection against the generation of dopa and other species that promote resistance to proteolysis might prove to be critical in preventing toxicity from oxidative stress in pathologies associated with protein deposition, such as atherosclerosis, Alzheimer's disease and Parkinson's disease.

Evidence for inactivation of cysteine proteases by reactive carbonyls via glycation of active site thiols.

Hyperglycaemia, triose phosphate decomposition and oxidation reactions generate reactive aldehydes in vivo. These compounds react non-enzymatically with protein side chains and N-terminal amino groups to give adducts and cross-links, and hence modified proteins. Previous studies have shown that free or protein-bound carbonyls inactivate glyceraldehyde-3-phosphate dehydrogenase with concomitant loss of thiol groups [Morgan, Dean and Davies (2002) Arch. Biochem. Biophys. 403, 259-269]. It was therefore hypothesized that modification of lysosomal cysteine proteases (and the structurally related enzyme papain) by free and protein-bound carbonyls may modulate the activity of these components of the cellular proteolytic machinery responsible for the removal of modified proteins and thereby contribute to a decreased removal of modified proteins from cells. It is shown that MGX (methylglyoxal), GO (glyoxal) and glycolaldehyde, but not hydroxyacetone and glucose, inhibit catB (cathepsin B), catL (cathepsin L) and catS (cathepsin S) activity in macrophage cell lysates, in a concentration-dependent manner. Protein-bound carbonyls produced similar inhibition with both cell lysates and intact macrophage cells. Inhibition was also observed with papain, with this paralleled by loss of the active site cysteine residue and formation of the adduct species S-carboxymethylcysteine, from GO, in a concentration-dependent manner. Inhibition of autolysis of papain by MGX, along with cross-link formation, was detected by SDS/PAGE. Treatment of papain and catS with the dialdehyde o-phthalaldehyde resulted in enzyme inactivation and an intra-molecular active site cysteine-lysine cross-link. These results demonstrate that reactive aldehydes inhibit cysteine proteases by modification of the active site cysteine residue. This process may contribute to the accumulation of modified proteins in tissues of people with diabetes and age-related pathologies, including atherosclerosis, cataract and Alzheimer's disease.

Biosynthesis and turnover of DOPA-containing proteins by human cells.

Protein-bound 3,4-dihydroxyphenylalanine (PB-DOPA) is a major product of hydroxyl radical attack on tyrosine residues of proteins. Levels of PB-DOPA in cells and tissues have been shown to be greatly elevated in age-related diseases. We demonstrate for the first time that l-DOPA (levodopa) can be biosynthetically incorporated into cell proteins by human cells (THP-1 monocytes and monocyte-derived macrophages). The DOPA-containing proteins generated were selectively visualized on PVDF membranes using a redox-cycling staining method. Many cell proteins contained DOPA and seemed to be synthesized as their full-length forms. The cellular removal of DOPA-containing proteins by THP-1 cells was by proteolysis involving both the proteasomal and the lysosomal systems. The rate of cellular proteolysis of DOPA-containing proteins increased at lower levels of DOPA incorporation but decreased at higher levels of DOPA incorporation. The decreased rate of degradation was accompanied by an increase in the activity of cathepsins B and L but the activity of cathepsin S increased only at lower levels of DOPA incorporation. These data raise the possibility that PB-DOPA could be generated in vivo from l-DOPA, which is the most widely used treatment for Parkinson disease.

Recent developments in the intracellular degradation of oxidized proteins.

The accumulation of oxidized proteins in cells and tissues is a feature of a number of age-related diseases and may also occur as a result of the aging process itself. In this article we review recent advances in our understanding of the cellular degradation of oxidized proteins directing our attention primarily to information which directly bears on the behavior of intact eukaryotic cells. We summarize new work on the key intracellular degradative machineries, proteasomes and lysosomes and examine evidence implicating an increase in protein hydrophobicity as the primary signal to the proteasome to initiate degradation. The data identifying the proteasome as the main route of degradation of oxidized proteins is examined, as well as recent data investigating changes in proteasome function after exposure of cells to oxidants and the altered catabolism of oxidized proteins in aging cells. Evidence for the cooperation between the lysosomal and proteasomal systems in the degradation of oxidized proteins is discussed. We conclude that the cellular catabolism of oxidized proteins may be a more complex process than it first appeared and suggest key issues that need to be resolved to improve our understanding of this important process.

Oxidative bioactivation of crotyl alcohol to the toxic endogenous aldehyde crotonaldehyde: association of protein carbonylation with toxicity in mouse hepatocytes.

Recent confirmation that the toxic unsaturated aldehyde crotonaldehyde (CA) contributes to protein damage during lipid peroxidation confers interest on the molecular actions of this substance. However, since a plethora of structurally related aldehydes form during membrane oxidation, clarifying the toxicological significance of individual products (e.g., CA) is challenging. To facilitate study of the mechanisms underlying CA toxicity, we explored the possibility that it can be formed enzymatically from an unsaturated precursor, crotyl alcohol. This is analogous to the way allyl alcohol is converted in vivo to its toxic oxidation product, acrolein. In kinetic studies, we found that crotyl alcohol was readily oxidized by equine liver alcohol dehydrogenase, with electrospray-mass spectrometry confirming that CA was the main product formed. Moreover, in mouse hepatocytes, crotyl alcohol produced marked time- and concentration-dependent cell killing as well as pronounced glutathione depletion. Both cytotoxicity and glutathione loss were abolished by the alcohol dehydrogenase inhibitor 4-methylpyrazole, indicating an oxidation product mediated these effects. In keeping with expectations that carbonyl-retaining Michael addition adducts would feature prominently during protein modification by CA, exposure to crotyl alcohol resulted in marked carbonylation of a wide range of cell proteins, an effect that was also abolished by 4-methylpyrazole. Damage to a subset of small proteins (e.g., 29, 32, 33 kDa) closely correlated with the severity of cell death. Collectively, these results demonstrate that crotyl alcohol is a useful tool for studying the biochemical and molecular events accompanying intracellular CA formation.