Affect Protein Levels Research Articles

Effect of Heat Treatment, Water and Vinegar Soaking on Protein and Phytic Acid Levels in Hemp Seed Meal

Hemp plants are notable for their climate resilience, and hempseed meal (HSM) is a potential high-protein feed for poultry. However, HSM has high levels of the antinutritional factor phytic acid (PA). This study aimed to evaluate the effects of heat and soaking treatments on the protein and PA content of HSM. HSM was obtained through cold pressing of whole hempseed and then subjected to heat treatment at 70°C for 24 hours. Soaking treatments involved water, water-vinegar mix, and vinegar for 1, 7, and 24 hours, followed by drying and analysis of PA and protein content. Results indicated that heating increased PA content without affecting protein levels. Soaking duration did not significantly alter protein content but did affect PA levels, with 24-hour soaking significantly increasing PA compared to 1-hour and 7-hour durations. The soaking material also influenced PA content: water soaking increased PA, while a 1-hour vinegar-water mix and 7-hour vinegar soaking significantly reduced PA. The highest PA concentration occurred with 24-hour water soaking. The protein content was highest with 7-hour vinegar soaking. In conclusion, acidic soaking solutions, particularly vinegar and vinegar-water mix, effectively reduced PA in HSM without protein loss.

Autoimmune hypothyroidism GWAS reveals independent autoimmune and thyroid-specific contributions and an inverse relation with cancer risk.

The high prevalence of autoimmune hypothyroidism (AIHT) - more than 5% in human populations - provides a unique opportunity to unlock the most complete picture to date of genetic loci that underlie systemic and organ-specific autoimmunity. Using a meta-analysis of 81,718 AIHT cases in FinnGen and the UK Biobank, we dissect associations along axes of thyroid dysfunction and autoimmunity. This largest-to-date scan of hypothyroidism identifies 418 independent associations (p < 5x10 - 8 ), more than half of which have not previously been documented in thyroid disease. In 48 of these, a protein-coding variant is the lead SNP or is highly correlated (r 2 > 0.95) with the lead SNP at the locus, including low-frequency coding variants at LAG3 , ZAP70 , TG, TNFSF11, IRF3, S1PR4, HABP2, ZNF429 as well as established variants at ADCY7, IFIH1 and TYK2 . The variants at LAG3 (P67T), ZAP70 (T155M), and TG (Q655X) are highly enriched in Finland and functional experiments in T-cells demonstrate that the ZAP70 :T155M allele reduces T-cell activation. By employing a large-scale scan of non-thyroid autoimmunity and a published meta-analysis of TSH levels, we use a Bayesian classifier to dissect the associated loci into distinct groupings and from this estimate, a significant proportion are involved in systemic (i.e., general to multiple autoimmune conditions) autoimmunity (34%) and another subset in thyroid-specific dysfunction (17%). By comparing these association results further to other common disease endpoints, we identify a noteworthy overlap with skin cancer, with 10% of AIHT loci showing a consistent but opposite pattern of association where alleles that increase the risk of hypothyroidism have protective effects for skin cancer. The association results, including genes encoding checkpoint inhibitors and other genes affecting protein levels of PD1, bolster the causal role of natural variation in autoimmunity influencing cancer outcomes.

Investigating the Correlation between Genotypes and Hepatic Protein Expression of CYP2C9, CYP2C19, CYP2D6, and CYP3A5 Using Postmortem Tissue from a Danish Population.

The cytochrome P450 (CYP) family of enzymes plays a central role in the metabolism of many drugs. CYP genes are highly polymorphic, which is known to affect protein levels, but for some low frequent CYP genotypes the correlation between genotype and CYP protein expression is less established. In this study, we determined the CYP2C9, CYP2C19, CYP2D6, and CYP3A5 genotypes of 250 Danish individuals included in a postmortem study. For 116 of the individuals, the hepatic CYP protein levels were investigated by a proteomics approach. Overall, we found the postmortem genetic and proteomic data to be in agreement with those of other studies performed on fresh hepatic tissue, showing the usability of postmortem hepatic tissue for this type of investigation. For less investigated genotypes, we could corroborate previously found results: 1) statistically significantly lower levels of hepatic CYP2C9 protein in individuals carrying the CYP2C9*3 variant compared with individuals with two wild type (wt) alleles; 2) comparable levels of CYP2C19 in CYP2C19*2/*17 and CYP2C19*1/*2 individuals; 3) reduced CYP2D6 protein levels in heterozygous individuals with the CYP2D6*3, CYP2D6*4, and CYP2D6*5 gene deletion variants; and 4) significantly lower levels of CYP3A5 protein in CYP3A5*3 homozygous individuals compared with individuals who were heterozygous for the CYP3A5*3 allele or homozygous individuals for the wt alleles. In conclusion, the use of postmortem tissue significantly increases the access to human specimens for research purposes, and postmortem proteomics can be used to investigate the link between CYP genotypes and hepatic protein expression. SIGNIFICANCE STATEMENT: In tissue samples from a large postmortem cohort (n = 250) we determined the CYP2C9, CYP2C19, CYP2D6, and CYP3A5 genotypes. Hepatic CYP protein levels were investigated in 116 individuals using a proteomics approach. For common genotypes, we found results similar to previous knowledge, pointing toward the usability of postmortem tissue. For the less investigated genotypes, we were able to corroborate genotype/protein expression correlations. It is a novel approach to use a large postmortem cohort to investigate genetic/protein expression correlations.

The glycogenolytic enzyme acid α-glucosidase is expressed in the bovine uterine endometrium.

Progesterone has been shown to stimulate glycogen catabolism in uterine epithelial cells. Acid α-glucosidase (GAA) is an enzyme that breaks down glycogen within lysosomes. We hypothesized that progesterone may stimulate glycogenolysis in the uterine epithelium via GAA. We found that GAA was more highly expressed in the stroma on Day 1 than on Day 11. However, GAA did not appear to differ in the epithelium on Days 1 and 11. Progesterone (0-10 μM) had no effect on the levels of the full-length inactive protein (110 kDa) or the cleaved (active) peptides present inside the lysosome (70 and 76 kDa) in immortalized bovine uterine epithelial (BUTE) cells. Furthermore, the activity of GAA did not differ between the BUTE cells treated with 10 μM progesterone or control. Overall, we confirmed that GAA is present in the cow endometrium and BUTE cells. However, progesterone did not affect protein levels or enzyme activity.

N-acetylcysteine modulates rotenone-induced mitochondrial Complex I dysfunction in THP-1 cells

Mitochondrial Complex I dysfunction and oxidative stress have been part of the pathophysiology of several diseases ranging from mitochondrial disease to chronic diseases such as diabetes, mood disorders and Parkinson’s Disease. Nonetheless, to investigate the potential of mitochondria-targeted therapeutic strategies for these conditions, there is a need further our understanding on how cells respond and adapt in the presence of Complex I dysfunction. In this study, we used low doses of rotenone, a classical inhibitor of mitochondrial complex I, to mimic peripheral mitochondrial dysfunction in THP-1 cells, a human monocytic cell line, and explored the effects of N-acetylcysteine on preventing this rotenone-induced mitochondrial dysfunction. Our results show that in THP-1 cells, rotenone exposure led to increases in mitochondrial superoxide, levels of cell-free mitochondrial DNA, and protein levels of the NDUFS7 subunit. N-acetylcysteine (NAC) pre-treatment ameliorated the rotenone-induced increase of cell-free mitochondrial DNA and NDUFS7 protein levels, but not mitochondrial superoxide. Furthermore, rotenone exposure did not affect protein levels of the NDUFV1 subunit but induced NDUFV1 glutathionylation. In summary, NAC may help to mitigate the effects of rotenone on Complex I and preserve the normal function of mitochondria in THP-1 cells.

Air pollution exposure increases ABCB1 and ASCT1 transporter levels in mouse cortex

Membrane transporters are important for maintaining brain homeostasis by regulating the passage of solutes into, out of, and within the brain. Growing evidence suggests neurotoxic effects of air pollution exposure and its contribution to neurodegenerative disorders, including Alzheimer’s disease (AD), yet limited knowledge is available on the exact cellular impacts of exposure. This study investigates how exposure to ubiquitous solid components of air pollution, ultrafine particles (UFPs), influence brain homeostasis by affecting protein levels of membrane transporters. Membrane transporters were quantified and compared in brain cortical samples of wild-type and the 5xFAD mouse model of AD in response to subacute exposure to inhaled UFPs. The cortical ASCT1 and ABCB1 transporter levels were elevated in wild-type and 5xFAD mice subjected to a 2-week UFP exposure paradigm, suggesting impairment of brain homeostatic mechanisms. This study provides new insight on the molecular mechanisms underlying adverse effects of air pollution on the brain.

Lactobacillus rhamnosus GG Promotes Intestinal Vitamin D Absorption by Upregulating Vitamin D Transporters in Senile Osteoporosis.

Intestinal absorption of vitamin D is an important way to improve the vitamin D level in senile osteoporosis (SOP). There is a link between oral probiotics and vitamin D, but the mechanism is still unclear. We aimed to evaluate whether Lactobacillus rhamnosus GG culture supernatant (LCS) can affect cholecalciferol absorption, transport, and hydroxylation in SOP, and explore underlying mechanisms. In the study, specific-pathogen-free SAMP6 mice were randomly divided into an experimental group administered undiluted LCS and a control group administered normal drinking water. Furthermore, levels of cholecalciferol absorption were compared between Caco-2 cells cultured with varying concentrations of cholecalciferol and stimulated with LCS or de Man, Rogosa, and Sharpe (MRS) broth (control). Similarly, LCS-stimulated HepG2 cells were compared with MRS-stimulated HepG2 cells. Finally, protein levels of VD transporters in small intestine tissues and Caco-2 cells, as well as vitamin D-binding protein and 25-hydroxylase in liver tissues and HepG2 cells, were detected by western blot. The results showed that plasma concentrations of cholecalciferol and 25OHD3 were higher in mice of the LCS group compared with the control group, and these values were positively correlated. With the addition of LCS, cholecalciferol uptake was increased with 0.5μM or 10μM cholecalciferol in the medium. Protein levels of CD36 and NPC1L1 were higher in the LCS group compared with the control group, while SR-BI protein was decreased, both in vitro and in vivo. In conclusion, LCS can promotes intestinal absorption cholecalciferol by affecting protein levels of VD transporters and improves 25OHD3 levels in SOP.

Tourniquet-induced lower limb ischemia/reperfusion reduces mitochondrial function by decreasing mitochondrial biogenesis in acute kidney injury in mice.

The mechanisms by which lower limb ischemia/reperfusion induces acute kidney injury (AKI) remain largely uncharacterized. We hypothesized that tourniquet‐induced lower limb ischemia/reperfusion (TILLIR) would inhibit mitochondrial function in the renal cortex. We used a murine model to show that TILLIR of the high thigh regions inflicted time‐dependent AKI as determined by renal function and histology. This effect was associated with decreased activities of mitochondrial complexes I, II, V and citrate synthase in the kidney cortex. Moreover, TILLIR reduced mRNA levels of a master regulator of mitochondrial biogenesis PGC‐1α, and its downstream genes NDUFS1 and ATP5o in the renal cortex. TILLIR also increased serum corticosterone concentrations. TILLIR did not significantly affect protein levels of the critical regulators of mitophagy PINK1 and PARK2, mitochondrial transport proteins Tom20 and Tom70, or heat‐shock protein 27. TILLIR had no significant effect on mitochondrial oxidative stress as determined by mitochondrial ability to generate reactive oxygen species, protein carbonylation, or protein levels of MnSOD and peroxiredoxin1. However, TILLIR inhibited classic autophagic flux by increasing p62 protein abundance and preventing the conversion of LC3‐I to LC3‐II. TILLIR increased phosphorylation of cytosolic and mitochondrial ERK1/2 and mitochondrial AKT1, as well as mitochondrial SGK1 activity. In conclusion, lower limb ischemia/reperfusion induces distal AKI by inhibiting mitochondrial function through reducing mitochondrial biogenesis. This AKI occurs without significantly affecting PINK1‐PARK2‐mediated mitophagy or mitochondrial oxidative stress in the kidney cortex.

Dynamic regulation of N6,2\u2032-O-dimethyladenosine (m6Am) in obesity

The prevalent m6Am mRNA cap modification was recently identified as a valid target for removal by the human obesity gene FTO along with the previously established m6A mRNA modification. However, the deposition and dynamics of m6Am in regulating obesity are unknown. Here, we investigate the liver m6A/m methylomes in mice fed on a high fat Western-diet and in ob/ob mice. We find that FTO levels are elevated in fat mice, and that genes which lost m6Am marking under obesity are overly downregulated, including the two fatty-acid-binding proteins FABP2, and FABP5. Furthermore, the cellular perturbation of FTO correspondingly affect protein levels of its targets. Notably, generally m6Am- but not m6A-methylated genes, are found to be highly enriched in metabolic processes. Finally, we deplete all m6A background via Mettl3 knockout, and unequivocally uncover the association of m6Am methylation with increased mRNA stability, translation efficiency, and higher protein expression. Together, these results strongly implicate a dynamic role for m6Am in obesity-related translation regulation.

Maize centromeric chromatin scales with changes in genome size.

Centromeres are defined by the location of Centromeric Histone H3 (CENP-A/CENH3) which interacts with DNA to define the locations and sizes of functional centromeres. An analysis of 26 maize genomes including 110 fully assembled centromeric regions revealed positive relationships between centromere size and genome size. These effects are independent of variation in the amounts of the major centromeric satellite sequence CentC. We also backcrossed known centromeres into two different lines with larger genomes and observed consistent increases in functional centromere sizes for multiple centromeres. Although changes in centromere size involve changes in bound CENH3, we could not mimic the effect by overexpressing CENH3 by threefold. Literature from other fields demonstrate that changes in genome size affect protein levels, organelle size and cell size. Our data demonstrate that centromere size is among these scalable features, and that multiple limiting factors together contribute to a stable centromere size equilibrium.

Stable Isotope Labeling of Amino Acids in Flies (SILAF) Reveals Differential Phosphorylation of Mitochondrial Proteins Upon Loss of OXPHOS Subunits

Drosophila melanogaster has been a workhorse of genetics and cell biology for more than a century. However, proteomic-based methods have been limited due to the complexity and dynamic range of the fly proteome and the lack of efficient labeling methods. Here, we advanced a chemically defined food source into direct stable-isotope labeling of amino acids in flies (SILAF). It allows for the rapid and cost-efficient generation of a large number of larvae or flies, with full incorporation of lysine-[13C6] after six labeling days. SILAF followed by fractionation and enrichment gave proteomic insights at a depth of 7196 proteins and 8451 phosphorylation sites, which substantiated metabolic regulation on enzymatic level. We applied SILAF to quantify the mitochondrial phosphoproteome of an early-stage leucine-rich PPR motif-containing protein (LRPPRC)-knockdown fly model of mitochondrial disease that almost exclusively affects protein levels of the oxidative phosphorylation (OXPHOS) system. While the mitochondrial compartment was hypo-phosphorylated, two conserved phosphosites on OXPHOS subunits NDUFB10 and NDUFA4 were significantly upregulated upon impaired OXPHOS function. The ease and versatility of the method actuate the fruit fly as an appealing model in proteomic and posttranslational modification studies, and it enlarges potential metabolic applications based on heavy amino acid diets.

Dynamic 3D proteomes reveal protein functional alterations at high resolution in situ

SUMMARYBiological processes are regulated by intermolecular interactions and chemical modifications that do not affect protein levels, thus escaping detection in classical proteomic screens. We demonstrate here that a global protein structural readout based on limited proteolysis-mass spectrometry (LiP-MS) detects many such functional alterations, simultaneously and in situ, in bacteria undergoing nutrient adaptation and in yeast responding to acute stress. The structural readout, visualized as structural barcodes, captured enzyme activity changes, phosphorylation, protein aggregation, and complex formation, with the resolution of individual regulated functional sites such as binding and active sites. Comparison with prior knowledge, including other ‘omics data, showed that LiP-MS detects many known functional alterations within well-studied pathways. It suggested distinct metabolite-protein interactions and enabled identification of a fructose-1,6-bisphosphate-based regulatory mechanism of glucose uptake in E. coli. The structural readout dramatically increases classical proteomics coverage, generates mechanistic hypotheses, and paves the way for in situ structural systems biology.

Preoperative Fasting and General Anaesthesia Alter the Plasma Proteome.

Background: Blood plasma collected at time of surgery is an excellent source of patient material for investigations into disease aetiology and for the discovery of novel biomarkers. Previous studies on limited sets of proteins and patients have indicated that pre-operative fasting and anaesthesia can affect protein levels, but this has not been investigated on a larger scale. These effects could produce erroneous results in case-control studies if samples are not carefully matched. Methods: The proximity extension assay (PEA) was used to characterize 983 unique proteins in a total of 327 patients diagnosed with ovarian cancer and 50 age-matched healthy women. The samples were collected either at time of initial diagnosis or before surgery under general anaesthesia. Results: 421 of the investigated proteins (42.8%) showed statistically significant differences in plasma abundance levels comparing samples collected at time of diagnosis or just before surgery under anaesthesia. Conclusions: The abundance levels of the plasma proteome in samples collected before incision, i.e., after short-time fasting and under general anaesthesia differs greatly from levels in samples from awake patients. This emphasizes the need for careful matching of the pre-analytical conditions of samples collected from controls to cases at time of surgery in the discovery as well as clinical use of protein biomarkers.

HSPA8 knock-down induces the accumulation of neurodegenerative disorder-associated proteins

Heat shock protein 70 family was demonstrated to play a critical role in protein homeostasis, a process profoundly impaired in neurodegenerative disorders. Neurodegenerative diseases are characterized by the accumulation of different kind of proteins and the formation of insoluble aggregates which are toxic for neurons. To explore the role of heat shock protein family 70 (in particular HSPA8 and HSPA1A) in the accumulation of proteins implied in neurodegeneration pathogenesis, in this study we verified in human SH-SY5Y neuroblastoma cells how HSPA8 or HSPA1A knock-down can affect protein levels of tau, superoxide dismutase 1 and α-synuclein. We found HSPA8 and HSPA1A reduction caused an increase of tau, superoxide dismutase 1 and α-synuclein protein levels. We also noticed HSPA8 knock-down increased α-synuclein oligomeric forms and mRNA expression. Our results suggest HSPA8 can play an important role in the homeostasis of tau, superoxide dismutase 1 and α-synuclein and in the balance between α-synuclein oligomeric and monomeric forms.

Abstract PR01: Three-hit model of Wilms’ tumor formation reveals immunogenic transcriptional subtypes

Abstract Wilms’ tumor is the most common kidney cancer in children. Despite advances in care, children with metastatic, anaplastic, or relapsed disease still fare poorly. Recent studies have uncovered novel types of driver mutations, including in microRNA processing genes such as DROSHA. However, there are no ways to rationally guide therapy based on mutation, as the mechanisms by which they cause cancer remain poorly defined. Thus, here we investigated how specific types of driver mutations affect gene and protein expression. To understand how these mutations drive Wilms’ tumor formation, we first recategorized known mutations and copy number changes using a new classification schema. We found four mutation classes to be mutually exclusive with each other: microRNA processing, MYCN-activating, chromatin remodeling, and RNA splicing. These mutations were not mutually exclusive with common mutations in kidney development genes or loss-of-heterozygosity/imprinting (LOH/LOI) of chr11p15. We propose that this mutational pattern implies a “three-hit” model, whereby 11p15 LOH/LOI and mutations impairing kidney development predispose to but are often not sufficient for Wilms’ tumor formation. A third mutation then transforms the transcriptome via microRNA processing, MYCN, chromatin remodeling, or splicing. To study how these “third hits” affect gene expression, we next performed gene set enrichment analysis. As expected, we found that microRNA impairment leads to overexpression of microRNA target genes, and MYCN activation drives MYC target genes. Interestingly, we also found that loss of microRNA processing correlated with expression of oxidative phosphorylation genes, which may reveal a metabolic dependency in these tumors. In addition, mutations affecting splicing led to high levels of interferon-stimulated genes. Thus, the abnormal RNA species generated by altered splicing appear to trigger the innate immune response that normally responds to viral RNA. Finally, we measured how these mutations affect protein levels using reverse-phase protein arrays. Strikingly, Wilms’ tumors with either anaplastic histology or microRNA processing mutations express high levels of immune-related markers such as PD-1, PD-L1, phospho-Stat3, and phospho-NF-kB. In summary, many Wilms’ tumors develop a total of three types of mutations: 11p15 LOH/LOI, kidney development impairment, and a third hit that reshapes the transcriptome in an oncogenic fashion. These “third hits” may affect microRNA processing, MYCN activity, chromatin remodeling, or RNA splicing, and each type of mutation has distinct effects on gene expression. In particular, mutations in microRNA processing cause aberrant overexpression of microRNA target genes, leading to metabolic reprogramming and increased immunogenicity. As a result, some Wilms’ tumor mutations have widespread effects on the transcriptome that may be susceptible to immune checkpoint blockade. This abstract is also being presented as Poster B05. Citation Format: Kenneth S. Chen, Kavita Desai, James F. Amatruda. Three-hit model of Wilms’ tumor formation reveals immunogenic transcriptional subtypes [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr PR01.

PENGARUH SUBSTITUSI TEPUNG SELPIS (SELUANG DAN PISANG) TERHADAP KADAR PROTEIN, KALSIUM, DAYA TERIMA DAN MUTU ORGANOLEPTIK COOKIES

Background: Insufficiency of nutrients intake such as protein and calcium may lead to the incidence of stunting in children under five years. In fact, Indonesia has many local foods like freshwater fish and local fruits which had abundant nutrients. Seluang is a typical Kalimantan freshwater fish contains high protein and calcium. The utilization of seluang fish flour and kepok banana flour as substitution in making cookies will increase the nutritional value of protein and calcium and may reduce the use of wheat flour.Objective: This study aims to analyse the effect of selpis (seluang and banana) flour substitution on protein, calcium, acceptability and organoleptic quality of cookies.Method: Experimental method with a completely randomized design (CRD) with five flour substitution treatments: 0%, 5%, 10%, 15%, 20% was used in this study. The protein content was tested by Kjeldahl method while the calcium content was tested by the AAS method.Results: Most of panelists preferred the original cookies (P1), characterized by yellowish in colour, fresh-baked aroma, slightly tough in texture and sweet-taste. The highest levels of protein and calcium were at P5 (20%) of 12.25% and 5.88 mg / 100 g.Conclusion: The substitution of seluang and kepok banana flours affect protein levels, calcium levels, acceptability (taste, color and aroma) of cookies but does not affect the texture of cookies.

The Effect of the Human MeCP2 gene on Drosophila melanogaster behavior and p53 inhibition as a model for Rett Syndrome

The purpose of this study was to explore the effect of introducing the human methyl CPG binding protein 2 gene (MeCP2) into the Drosophila melanogaster genome, examine how it would affect protein levels of p53, and test how inhibiting p53 with pifithrin-alpha would affect behavioral and physical patterns in fruit flies. One goal was to observe if the symptoms of Rett Syndrome, a neurodegenerative disease in humans, are reflected in Drosophila melanogaster. This was achieved by differentiating the behavior and physical aspects of wild-type flies from flies expressing the full-length MeCP2 gene and the mutated MeCP2 gene (R106W). Differences in eye structure, climbing (the use of locomotor skills), geotaxis, as well as differences in the flies’ abilities to fly were analyzed. In addition, after these series of tests, the MeCP2 protein and the p53 protein, endogenous to Drosophila, were extracted and their levels were compared in each of the populations of flies. Then, after the Drosophila consumed pifithrin-alpha hydrobromide, their behavior, physical appearance, and protein levels were again monitored. After conducting these experiments, the effects of full-length and mutant MeCP2 on Drosophila melanogaster’s psychomotor skills were analyzed, along with their effects on how the treatment of pifithrin-alpha affected the level of MeCP2 and p53 in the various populations of flies. In conclusion, some of the Rett Syndrome symptoms were recapitulated in Drosophila, and a subset of those were partially ameliorated by the introduction of pifithrin-alpha.

Activation of adenosine receptors modulates the efflux transporters in brain capillaries and restores the anticonvulsant effect of carbamazepine in carbamazepine resistant rats developed by window-pentylenetetrazole kindling

Up-regulation of efflux transporters in brain capillaries may lead to the decreased therapeutic efficacy of antiepileptic drugs in patients with Drug Resistant Epilepsy. Adenosine receptor activation in brain capillaries can modulate blood-brain barrier permeability by decreasing the protein levels and function of efflux transporters. Therefore, we aimed to investigate whether the activation of adenosine receptors improves convulsions outcome in carbamazepine (CBZ) resistant animals and modulates the protein levels of efflux transporters (P-GP, MRP1, MRP2) in brain capillaries. We employed the window-pentylenetetrazol (PTZ) kindling model to develop CBZ resistant rats by CBZ administration during the post-kindling phase, and tested if these animals displayed subsequent resistance to other antiepileptic drugs. Crucially, we investigated if the administration of a broad-spectrum adenosine agonist (NECA) improves convulsions control in CBZ resistant rats. Of potential therapeutic relevance, in CBZ resistant rats NECA restored the anticonvulsant effect of CBZ. We also evaluated how the resistance to CBZ and the activation of adenosine receptors with NECA affect protein levels of efflux transporters in brain capillaries, as quantified by western blot. While CBZ resistance was associated with the up-regulation of both P-GP/MRP2 in brain capillaries, with the administration of NECA in CBZ resistant rats, we observed a decrease of P-GP and an increase of MRP2 levels, in brain capillaries. Since the activation of adenosine receptors improves the outcome of convulsions probably through the modulation of the efflux transporters protein levels in brain capillaries, adenosine agonists could be useful as an adjunct therapy for the control of Drug Resistant Epilepsy.

Functional study and pathogenicity classification of PRRT2 missense variants in PRRT2-related disorders.

Aims PRRT2 variants are associated with various paroxysmal disorders. To date, more than 90 PRRT2 variants have been reported in PRRT2‐related disorders. Lack of functional study in majority of missense variants makes their pathogenicity uncertain. We aim to evaluate the clinical significance of PRRT2 missense variants by performing in vitro experiments.MethodsWe systematically reviewed PRRT2‐related disorders and summarized reported PRRT2 missense variants. Protein expression and subcellular localization of mutant PRRT2 were investigated in mammal cells. American College of Medical Genetics and Genomics (ACMG) guidelines were used to analyze the pathogenicity of PRRT2 missense variants.ResultsA total of 29 PRRT2 missense variants were identified in PRRT2‐related disorders. Ten variants were observed to affect both subcellular localization and protein level, three variants only affect membrane localization, and two variants only affect protein level. According to ACMG guidelines, 15 variants were finally classified as “likely pathogenic”, three as “benign”, three as “likely benign”, and eight as “uncertain significance” variants. The likely pathogenic variants were concentrated in the C‐terminal of PRRT2.ConclusionsThe pathogenicity of eight uncertain significance variants needs further investigation. C‐terminal of PRRT2 is crucial for its physiological function.

Increased cysteine sulfonation of Complex III with destruction of heme c mediates cardiac reperfusion injury via impairing catalytic function and enhancing FADH2‐linked superoxide production

One consequence of myocardial ischemia-reperfusion injury (I/R) is oxidative damage, which can cause mitochondrial dysfunction. Such I/R-induced mitochondrial dysfunction results in impaired state 3 respiration and overproduction of superoxide, which can potentially induce protein cysteine sulfonation (PrSO3H) in mitochondria. Herein we employed UV/VIS spectroscopy and LC-MS/MS analysis to study the mechanism of I/R-mediated oxidative injury of complex III in mitochondria from rat hearts subjected to 30-min of ischemia and 24-h of reperfusion in vivo. In the I/R region, the activity of complex III was decreased by 31.0±4.1 % (n=7, p<0.001). Visible spectroscopic and immunoblotting analysis indicated that I/R mediated destruction of heme c (c-type heme) without affecting protein level of cytochrome c (cyt c) in the mitochondria. I/R thus mediates dysfunction of cyt c via heme destruction. However, no significant impairment of complex III activity and heme c destruction was observed in mitochondria isolated from the risk region of rat heart subjected to 30 min ischemia (no reperfusion) in vivo despite a decreased state 3 respiration. LC-MS/MS analysis showed enhanced formation of PrSO3H, which was consistent with mitochondrial redox state following I/R (EPR measurement of cyclic hydroxylamine oxidation). Mitochondria from I/R regions had intensely increased oxidative modification with sulfonation of cysteine (at C236) of 2Fe-2S cluster of Rieske iron-sulfur protein (uqcrfs1), which would impair the main electron transfer pathway controlled by the Q-cycle mechanism of complex III. LC-MS/MS analysis also indicated that I/R intensely increased complex III PrSO3H at the subunits of hinge protein, cytochrome c1, core 1 and core 2. Carbamidomethylated C122/C125 of the cytochrome c1 via alkylating complex III was exclusively detected in the post-ischemic mitochondria by LC-MS/MS, indicating I/R mediates cleavage of thioether bonds between heme c1 and apocytochrome c1. FADH2-linked ·O2− generation by mitochondrial SCR (supercomplex hosting complex II and complex III) was induced by succinate and evaluated by EPR spin trapping with DEPMPO. FADH2-linked ·O2− generation by normal mitochondrial SCR was minimized in the presence of a functional cyt c. Whereas, FADH2-linked ·O2− generation by the post-ischemic mitochondrial SCR was intensely increased by 123% in the presence of heme-depleted cyt c, and further enhanced by 227% under the conditions of increasing pO2 in vitro (from 0.23 mM O2 to 0.8 mM O2), suggesting that physiologic conditions of hyperoxygenation during reperfusion with heme-depleted cyt c mediated overproduction of FADH2-linked ·O2− by the post-ischemic mitochondria. In conclusion, reperfusion-induced PrSO3H with heme depletion of cyt c controls oxidative injury of the complex III, and aggravates mitochondrial dysfunction via overproducing ·O2− in the post-ischemic heart. Preservation of mitochondrial function and ultimately the salvage of ischemic tissue may necessitate lessening of PrSO3H and preservation of functional integrity of heme groups in mitochondria. Support or Funding Information NIH RO1HL083237 Enhancement of FADH2-linked superoxide generation by post-ischemic mitochondrial SCR (C vs B) in the presence of post-ischemic cyt c (D vs B) and hyperoxygenation (E vs D). This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.