The Mechanism of NEDD8 Activation of CUL5 Ubiquitin E3 Ligases.

Cullin RING E3 ligases (CRLs) ubiquitylate hundreds of important cellular substrates. Here we have assembled and purified the Ankyrin repeat and SOCS Box protein 9 CUL5 RBX2 ligase (ASB9-CRL) in vitro and show how it ubiquitylates one of its substrates, CKB. CRLs occasionally collaborate with RING between RING E3 ligases (RBRLs), and indeed, mass spectrometry analysis showed that CKB is specifically ubiquitylated by the ASB9-CRL-ARIH2-UBE2L3 complex. Addition of other E2s such as UBE2R1 or UBE2D2 contributes to polyubiquitylation but does not alter the sites of CKB ubiquitylation. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis revealed that CUL5 neddylation allosterically exposes its ARIH2 binding site, promoting high-affinity binding, and it also sequesters the NEDD8 E2 (UBE2F) binding site on RBX2. Once bound, ARIH2 helices near the Ariadne domain active site are exposed, presumably relieving its autoinhibition. These results allow us to propose a model of how neddylation activates ASB-CRLs to ubiquitylate their substrates.

Strategies to improve postpartum glucose screening rates are needed.

AIMS: Women with gestational diabetes mellitus (GDM) require postpartum glucose screening, as they have a 70% lifetime risk of developing Type 2 diabetes mellitus. However, less than half complete postpartum screening. METHODS: We conducted a retrospective chart review of patients who delivered at our institution from 2001 to 2019. Inclusion criteria were patients with gestational diabetes who were at least 18 years old and had delivered an infant at >24 weeks of gestation. Our primary outcome was completion of postpartum gestational diabetes screening. RESULTS: The majority of patients (62%) did not complete screening. After adjusted risk ratio analyses, the only variables that remained significantly associated with an increased likelihood of completing screening were Asian race and having prenatal care at one particular community health center, which served a predominantly Asian population. CONCLUSIONS: This community health center protocol for scheduling patients with GDM that complied with recommendations for postpartum care, indicating that evidence-based methods can improve maternal health.

Symbiont abundance can affect host plant population dynamics.

PREMISE OF THE STUDY: Symbioses are almost universal, but little is known about how symbiont abundance can affect host performance. Many orchids undergo vegetative dormancy and frequent and protracted dormancy have been associated with population declines. If mycorrhizal fungi affect host plant performance, those effects are likely to alter patterns of vegetative dormancy. The goal of this study was to determine whether the abundance of mycorrhizal fungi is related to the likelihood of entering dormancy and whether fungal abundance varied with dormancy duration in the federally listed threatened orchid Isotria medeoloides. METHODS: We studied three populations of the threatened North American terrestrial orchid Isotria medeoloides using long-term emergence data and evaluated the relationship between the abundance of associated mycorrhizal fungi (Russulaceae) and orchid dormancy and emergence. Mycorrhizal fungi in soil adjacent to orchids were quantified in two ways. First, ectomycorrhizal (ECM) fungi on adjacent root tips were identified using DNA sequencing to determine their phylogenetic relationship to fungi that are known to form mycorrhizae with I. medeoloides. Second, we extracted DNA from soil samples and used quantitative real-time PCR to estimate the abundance of Russulaceae hyphae adjacent to each orchid. KEY RESULTS: We found that the abundance of Russulaceae, both in the soil and on nearby ECM root tips, was significantly related to orchid prior emergence. Both abundance and prior emergence history were predictive of future emergence. CONCLUSIONS: These results suggest that the abundance of mycorrhizal fungi can influence orchid population dynamics and is an essential component of orchid conservation.

Mitochondrial dysfunction and complications associated with diabetes.

BACKGROUND: Diabetes is a metabolic syndrome that results in chronically increased blood glucose (hyperglycaemia) due to defects either in insulin secretion consequent to the loss of beta cells in the pancreas (type 1) or to loss of insulin sensitivity in target organs in the presence of normal insulin secretion (type 2). Long term hyperglycaemia can lead to a number of serious health-threatening pathologies, or complications, especially in the kidney, heart, retina and peripheral nervous system. SCOPE OF REVIEW: Here we summarise the current literature on the role of the mitochondria in complications associated with diabetes, and the limitations and potential of rodent models to explore new modalities to limit complication severity. MAJOR CONCLUSIONS: Prolonged hyperglycaemia results in perturbation of catabolic pathways and in an over-production of ROS by the mitochondria, which in turn may play a role in the development of diabetic complications. Furthermore, current models don't offer a comprehensive recapitulation of these complications. GENERAL SIGNIFICANCE: The onset of complications associated with type 1 diabetes can be varied, even with tightly controlled blood glucose levels. The potential role of inherited, mild mitochondrial dysfunction in accelerating diabetic complications, both in type 1 and 2 diabetes, remains unexplored. This article is part of a Special Issue entitled Frontiers of Mitochondrial Research.

Increased Diabetes Complications in a Mouse Model of Oxidative Stress Due to 'Mismatched' Mitochondrial DNA.

Associations between chronic diabetes complications and mitochondrial dysfunction represent a subject of major importance, given the diabetes pandemic and high personal and socioeconomic costs of diabetes and its complications. Modelling diabetes complications in inbred laboratory animals is challenging due to incomplete recapitulation of human features, but offer mechanistic insights and preclinical testing. As mitochondrial-based oxidative stress is implicated in human diabetic complications, herein we evaluate diabetes in a unique mouse model that harbors a mitochondrial DNA from a divergent mouse species (the 'xenomitochondrial mouse'), which has mild mitochondrial dysfunction and increased oxidative stress. We use the streptozotocin-induced diabetes model with insulin supplementation, with 20-weeks diabetes. We compare C57BL/6 mice and the 'xenomitochondrial' mouse, with measures of heart and kidney function, histology, and skin oxidative stress markers. Compared to C57BL/6 mice, the xenomitochondrial mouse has increased diabetic heart and kidney damage, with cardiac dysfunction, and increased cardiac and renal fibrosis. Our results show that mitochondrial oxidative stress consequent to divergent mtDNA can worsen diabetes complications. This has implications for novel therapeutics to counter diabetes complications, and for genetic studies of risk, as mtDNA genotypes may contribute to clinical outcomes.

The Social Context of Unhealthy Alcohol Use Among Emergency Department Patients: A Cross Sectional Study.

BACKGROUND: Housing status and additional social determinants of health are important data for clinicians and policy makers to design and implement effective interventions for emergency department (ED) patients with unhealthy alcohol use (UAU). METHODS: We surveyed patients in an urban, safety-net ED from June to August 2018. UAU was assessed by a validated single-item screening question endorsed by the National Institute on Alcohol Abuse and Alcoholism. Housing status was assessed using items validated for housing stability. RESULTS: Seven hundred fifty-eight patients completed the survey (60% response rate), and 296 (39%; 95% confidence interval: 36%-43%) reported UAU. Patients with and without UAU had the same rates of ED visits (median 2, interquartile range: 1-4; P = 0.69) and hospitalizations (median 0, interquartile range: 0-0; P = 0.31) in the 12 months before index visit. Patients with UAU were more likely to lack stable housing compared to patients without UAU (69% vs 59%; P = 0.006). Illicit drug use and prescription drug misuse was more common in patients with UAU compared to those without UAU (29% vs 14%, P < 0.001; and 18% vs 10%; P < 0.001, respectively). Only 60 (20.3%) of the 296 patients with UAU had a documented diagnosis of UAU in the medical record. CONCLUSIONS: UAU is common in the general ED patient population and usually not clinically recognized. Patients with UAU have high rates of homelessness and co-occurring substance use. Future studies should consider strategies to incorporate social determinants of health and harm reduction treatments into ED-based interventions for UAU.

Clinical features and long-term follow-up of 70 cases of canine idiopathic eosinophilic lung disease.

BACKGROUND: Canine idiopathic eosinophilic lung disease (ELD) is sparsely documented in the literature. METHODS: Clinical presentation and outcome of dogs diagnosed with ELD (eosinophilic bronchitis or eosinophilic bronchopneumonia) were reviewed. Subgroups were made based on chronicity of clinical signs and findings of thoracic imaging: NCI (no changes in thoracic imaging), BRON (bronchial/peribronchial pattern), INT (bronchointerstitial/interstitial/alveolar). RESULTS: Seventy cases were included. There were more young to adult, crossbreed and female dogs. Compared with the other two groups NCI dogs showed lower bronchoalveolar lavage fluid eosinophilic pleocytosis and absence of circulating eosinophilia, bronchiectasis or death due to respiratory disease. All dogs responded clinically to corticosteroids. Median treatment duration was four months. Remission (no clinical signs after treatment discontinuation for >one month) and long-term remission (>six months) was achieved in 60 per cent, and 51 per cent of patients, respectively. Relapse occurred in 26 per cent of cases after remission but was rare (3 per cent) after long-term remission. The one-year, two-year and four-year survival to death due to respiratory disease was 98 per cent, 97 per cent and 91 per cent, respectively. CONCLUSION: Prognosis and initial clinical response for ELD was generally good although achievement of long-term remission was only seen in 51 per cent of dogs. Different outcomes based on chronicity of signs, corticosteroid dose, thoracic imaging abnormalities and other clinical variables were not appreciated.

Survival of activated myofibroblasts in canine myxomatous mitral valve disease and the role of apoptosis.

Myxomatous mitral valve disease (MMVD) is the single most important acquired cardiovascular disease of the dog. Much is known about the cellular changes and the contribution of activated myofibroblasts (valve interstitial cells (aVICs) to the valve extra-cellular matrix remodelling characteristic of the disease. However, little is known on how aVIC survival might contribute to disease pathogenesis. This study examined the temporal (disease severity-dependent) and spatial distribution of aVICs in MMVD valves, the expression of a range of apoptosis-related genes in cultured VICs from both normal (quiescent VIC (qVIC) and diseased (aVIC) valves, and the differential effects of doxorubicin treatment, as a trigger of apoptosis, on expression of the same genes. Activated myofibroblasts were identified in normal valves at the valve base only (the area closest to the annulus), and then became more numerous and apparent along the valve length as the disease progressed, with evidence of cell survival at the valve base. There were no significant differences in basal gene expression comparing qVICs and aVICs for CASP3, FAS, BID, BAX, BCL2, CASP8, DDIAS, XIAP and BIRC5. After doxorubicin treatment (2 mM) for 8 h there was significant difference (P < .05) in the expression of BID, BCL2, DDIAS, and CASP8, but when assessed for interactions using a mixed model ANOVA only CASP8 was significantly different because of treatment (P < .05). These data suggest aVIC survival in MMVD valves may be a consequence of heightened resistance of aVICs to apoptosis, but would require confirmation examining expression of the relevant proteins.

Restored degradation of the Alzheimer's amyloid-beta peptide by targeting amyloid formation.

Accumulation of neurotoxic amyloid-beta (Abeta) is central to the pathology of Alzheimer's disease (AD). Elucidating the mechanisms of Abeta accumulation will therefore expedite the development of Abeta-targeting AD therapeutics. We examined activity of an Abeta-degrading protease (matrix metalloprotease 2) to investigate whether biochemical factors consistent with conditions in the AD brain contribute to Abeta accumulation by altering Abeta sensitivity to proteolytic degradation. An Abeta amino acid mutation found in familial AD, Abeta interactions with zinc (Zn), and increased Abeta hydrophobicity all strongly prevented Abeta degradation. Consistent to all of these factors is the promotion of specific Abeta aggregates where the protease cleavage site, confirmed by mass spectrometry, is inaccessible within an amyloid structure. These data indicate decreased degradation due to amyloid formation initiates Abeta accumulation by preventing normal protease activity. Zn also prevented Abeta degradation by the proteases neprilysin and insulin degrading enzyme. Treating Zn-induced Abeta amyloid with the metal-protein attenuating compound clioquinol reversed amyloid formation and restored the peptide's sensitivity to degradation by matrix metalloprotease 2. This provides new data indicating that therapeutic compounds designed to modulate Abeta-metal interactions can inhibit Abeta accumulation by restoring the catalytic potential of Abeta-degrading proteases.

Evaluation of canine 2D cell cultures as models of myxomatous mitral valve degeneration.

The utility of cells cultured from the mitral valve as models of myxomatous diseases needs to be properly validated. In this study valve interstitial cells (VICs) and valve endothelial cells (VECs) were cultured from normal and diseased canine mitral valves in 2% (v/v) or 10% FBS media, in the presence of TGFß1, 2 and 3, the TGFß RI kinase inhibitor SB431542 and TGFß neutralising antibodies, 5HT and the 5HT2RB antagonist LY272015. Cultures were examined by morphology, transcriptomic profiling, protein expression of the cell specific markers αSMA and SM22α (VICs), and CD31 (VECs), deposition of proteoglycans (PG), the PG versican, and the TGFßs themselves. VECs derived from normal valves were CD31+/αSMA-, but those from diseased valves were αSMA+, indicating endothelial-to-mesenchymal (EndoMT) transition had occurred. The TGFßs induced EndoMT in normal VECs, and this was abolished by SB431542, with significant changes in αSMA, CD31 and HAS2 expression (P<0.05). Normal VICs cultured in 10% FBS media were αSMA+ (activated myofibroblast (disease) phenotype), but were αSMA- when grown in 2% FBS. VICs from diseased dogs were αSMA+ in 2% FBS (retention of the activated myofibroblast disease phenotype), with significantly increased TGFß1 expression (P<0.05) compared to normal cells. Treatment of normal and diseased VICs with the TGFßs significantly increased expression of αSMA, SM22α, versican, the TGFßs themselves, and deposition of PGs (P<0.05), with TGFß1 being the most potent activator. These effects were either abolished or markedly reduced by SB431542 and a pan-TGFß neutralizing antibody (P<0.05). SB431542 also markedly reduced αSMA expression in VICs from diseased valves, but 5HT and LY272015 had no effect on VIC phenotype. Transcriptomic profiling identified clear differences in gene expression for the different conditions and treatments that partially matched that seen in native diseased valve tissue, including changes in expression of ACTA2 (αSMA), 5HTR2B, TAGLN (SM22α) and MYH10 (SMemb), gene ontology terms and canonical signalling pathways. Normal and diseased VICs and normal VECs from canine mitral valves can be successfully grown in culture with retention of phenotype, which can be manipulated using TGFß1 and the TGFß RI kinase inhibitor SB431542. This optimized cell system can now be used to model MMVD to elucidate disease mechanisms and identify key regulators of disease progression.

Vascular distribution of nitric oxide synthase and vasodilation in the Australian lungfish, Neoceratodus forsteri.

The presence of nitric oxide synthase (NOS) and role of nitric oxide (NO) in vascular regulation was investigated in the Australian lungfish, Neoceratodus forsteri. No evidence was found for NOS in the endothelium of large and small blood vessels following processing for NADPH-diaphorase histochemistry. However, both NADPH-diaphorase histochemistry and neural NOS immunohistochemistry demonstrated a sparse network of nitrergic nerves in the dorsal aorta, hepatic artery, and branchial arteries, but there were no nitrergic nerves in small blood vessels in tissues. In contrast, nitrergic nerves were found in non-vascular tissues of the lung, gut and kidney. Dual-wire myography was used to determine if NO signalling occurred in the branchial artery of N. forsteri. Both SNP and SIN-1 had no effect on the pre-constricted branchial artery, but the particulate guanylyl cyclase (GC) activator, C-type natriuretic peptide, always caused vasodilation. Nicotine mediated a dilation that was not inhibited by the soluble GC inhibitor, ODQ, or the NOS inhibitor, L-NNA, but was blocked by the cyclooxygenase inhibitor, indomethacin. These data suggest that NO control of the branchial artery is lacking, but that prostaglandins could be endothelial relaxing factors in the vasculature of lungfish.

Linker histone H1 binds to disease associated amyloid-like fibrils.

Alzheimer's disease (AD) and Parkinson's disease (PD) are the two most prevalent neurodegenerative diseases of the central nervous system. These two diseases share a common feature in that a normally soluble peptide (amyloid-beta) or protein (alpha-synuclein) aggregates into an ordered fibrillar structure. As well as structural similarities observed between fibrillar aggregates related to these diseases, common pathological processes of increased oxidative injury, excitotoxicity and altered cell cycle are also evident. It was the aim of this study to identify novel interacting proteins to the amyloid-like motif and therefore identify common potential pathways between neurodegenerative diseases that share biophysical properties common to classical amyloid fibrils. Optimal ageing of recombinant proteins to form amyloid-like fibrils was determined by electron microscopy, Congo red birefringement and photo-induced cross-linking. Using pull-down assays the strongest detected interacting protein to the amyloid-like motifs of amyloid-beta, alpha-synuclein and lysozyme was identified as histone H1. The interaction with the amyloid-like motif was confirmed by techniques including surface plasmon resonance and immunohistochemistry. Histone H1 is known to be an integral part of chromatin within the nucleus, with a primary role of binding DNA that enters and exits from the nucleosome, and facilitating the shift in equilibrium of chromatin towards a more condensed form. However, phosphorylated histone H1 is predominantly present in the cytoplasm and as yet the functional significance of this translocation is unknown. This study also found that histone H1 is localised within the cytoplasm of neurons and astrocytes from areas affected by disease as well as amyloid plaques, supporting the hypothesis that histone H1 favoured binding to an ordered fibrillar motif. We conclude that the binding of histone H1 to a general amyloid-like motif indicates that histone H1 may play an important common role in diseases associated with amyloid-like fibrils.

Evaluation of eicosanoid concentrations in stored units of canine packed red blood cells.

OBJECTIVE To evaluate eicosanoid concentrations in freshly prepared canine packed RBCs (PRBCs) and to assess changes in eicosanoid concentrations in PRBC units over time during storage and under transfusion conditions. DESIGN Prospective study. SAMPLE 25 plasma samples from 14 healthy Greyhounds. PROCEDURES Plasma samples were obtained during PRBC preparation (donation samples), and the PRBC units were then stored at 4°C until used for transfusion (≤ 21 days later; n = 17) or mock transfusion if expired (22 to 24 days later; 8). Immediately prior to use, 100 mL of saline (0.9% NaCl) solution was added to each unit and a pretransfusion sample was collected. A posttransfusion sample was collected after transfusion or mock transfusion. Concentrations of arachidonic acid, prostaglandin (PG) F2α, PGE2, PGD2, thromboxane B2, 6-keto-PGF1α, and leukotriene B4 were measured by liquid chromatography-mass spectrometry and analyzed statistically. RESULTS Median arachidonic acid concentration was significantly decreased in posttransfusion samples, compared with the concentration in donation samples. Median PGF2α, 6-keto-PGF1α, and leukotriene B4 concentrations were significantly increased in pretransfusion samples, compared with those in donation samples. Median PGF2α, thromboxane B2, and 6-keto-PGF1α concentrations were significantly increased in posttransfusion samples, compared with those in pretransfusion samples. Duration of PRBC storage had significant associations with pretransfusion and posttransfusion arachidonic acid and thromboxane B2 concentrations. CONCLUSIONS AND CLINICAL RELEVANCE Concentrations of several proinflammatory eicosanoids increased in PRBC units during storage, transfusion, or both. Accumulation of these products could potentially contribute to adverse transfusion reactions, and investigation of the potential association between eicosanoid concentrations in PRBCs and the incidence of transfusion reactions in dogs is warranted.

Copper-dependent inhibition of human cytochrome c oxidase by a dimeric conformer of amyloid-beta1-42.

In studies of Alzheimer's disease pathogenesis there is an increasing focus on mechanisms of intracellular amyloid-beta (Abeta) generation and toxicity. Here we investigated the inhibitory potential of the 42 amino acid Abeta peptide (Abeta1-42) on activity of electron transport chain enzyme complexes in human mitochondria. We found that synthetic Abeta1-42 specifically inhibited the terminal complex cytochrome c oxidase (COX) in a dose-dependent manner that was dependent on the presence of Cu2+ and specific "aging" of the Abeta1-42 solution. Maximal COX inhibition occurred when using Abeta1-42 solutions aged for 3-6 h at 30 degrees C. The level of Abeta1-42-mediated COX inhibition increased with aging time up to approximately 6 h and then declined progressively with continued aging to 48 h. Photo-induced cross-linking of unmodified proteins followed by SDS-PAGE analysis revealed dimeric Abeta as the only Abeta species to provide significant temporal correlation with the observed COX inhibition. Analysis of brain and liver from an Alzheimer's model mouse (Tg2576) revealed abundant Abeta immunoreactivity within the brain mitochondria fraction. Our data indicate that endogenous Abeta is associated with brain mitochondria and that Abeta1-42, possibly in its dimeric conformation, is a potent inhibitor of COX, but only when in the presence of Cu2+. We conclude that Cu2+-dependent Abeta-mediated inhibition of COX may be an important contributor to the neurodegeneration process in Alzheimer's disease.

LBW and SGA Impact Longitudinal Growth and Nutritional Status of Filipino Infants.

CLINICAL TRIAL REGISTRATION: NCT00486863.

Attempts to control unwanted thoughts in the night: development of the thought control questionnaire-insomnia revised (TCQI-R).

The attempted control of intrusive, uncontrollable thoughts has been implicated in the maintenance of a range of psychological disorders. The current paper describes the refinement of the Thought Control Questionnaire Insomnia (TCQI; Behav. Cogn. Psychoth. 29 (2001)) through its administration to a sample (n=385) including good sleepers and individuals with insomnia. Several items with poor psychometric properties were discarded, resulting in a 35-item revised TCQI. Factor analysis revealed six factors; aggressive suppression, cognitive distraction, reappraisal, social avoidance, behavioural distraction, and worry. The attempted management of unwanted thoughts was compared across individuals with insomnia and good sleepers, and the impact of these strategies on sleep quality, anxiety and depression was investigated. With the exception of cognitive distraction, individuals with insomnia, relative to good sleepers, more frequently used every thought control strategy. The strategies of aggressive suppression and worry, in particular, appeared to be unhelpful, with the use of these strategies predicting sleep impairment, anxiety and depression. The strategy of cognitive distraction appeared to be helpful, with the use of this strategy predicting better sleep quality.

Gender Differences in Scholarly Productivity Within Academic Gynecologic Oncology Departments.

OBJECTIVE: To estimate whether there is a gender difference in scholarly productivity among academic gynecologic oncologists. METHODS: In this cross-sectional study, the academic rank and gender of gynecologic oncology faculty in the United States were determined from online residency and fellowship directories and departmental web sites. Each individual's h-index and years of publication were determined from Scopus (a citation database of peer-reviewed literature). The h-index is a quantification of an author's scholarly productivity that combines the number of publications with the number of times the publications have been cited. We generated descriptive statistics and compared rank, gender, and productivity scores. RESULTS: Five hundred seven academic faculty within 137 U.S. teaching programs were identified. Of these, 215 (42%) were female and 292 (58%) were male. Men had significantly higher median h-indices than women, 16 compared with 8, respectively (P<.001). Women were more likely to be of junior academic rank with 63% of assistant professors being female compared with 20% of full professors. When stratifying h-indices by gender and academic rank, men had significantly higher h-indices at the assistant professor level (7 compared with 5, P<.001); however, this difference disappeared at the higher ranks. Stratifying by the years of active publication, there was no significant difference between genders. CONCLUSION: Female gynecologic oncologists at the assistant professor level had lower scholarly productivity than men; however, at higher academic ranks, they equaled their male counterparts. Women were more junior in rank, had published for fewer years, and were underrepresented in leadership positions. LEVEL OF EVIDENCE: III.

Health disparity curriculum at the Warren Alpert Medical School of Brown University.

There is increasing recognition that, in addition to acquiring knowledge of basic sciences and clinical skills, medical students must also gain an understanding of health disparities, and develop a defined skill set to address these inequalities. There are few descriptions in the literature of a systematic, longitudinal curriculum in health disparities. Using Kern's six-step approach to curriculum development along with principles of experiential and active learning, student champions and the Office of Medical Education developed a multimodal health disparities curriculum. This curriculum includes required experiences for medical students in the 1st, 2nd and 3rd year, along with elective experiences throughout medical school. Students are examined on their knowledge, skills and attitudes towards health disparities prior to graduation. It is our hope this curriculum empowers students with the knowledge, skills and attitudes to care for patients while helping patients navigate the socioeconomic and cultural issues that may affect their health.

Oxidative phosphorylation measurement in cell lines and tissues.

Mitochondrial oxidative phosphorylation (OXPHOS) dysfunction is implicated in a growing spectrum of diseases, from neurodegeneration to cancer. Where tissues or transformed cells are available, respirometry and enzymology allow a sophisticated analysis of OXPHOS with modest-cost equipment. The isolation of organelle fractions is also invaluable for determining association of proteins of interest. Here we revisit and consolidate methods to measure whole cell mitochondrial ATP synthesis, respiration, isolation of mitochondria from cultured cells and tissues, and OXPHOS enzymology. We also explain common pitfalls, guide optimisation of the methods for new users, and provide full laboratory protocols in Supplementary materials.

Copper-dependent inhibition of cytochrome c oxidase by Abeta(1-42) requires reduced methionine at residue 35 of the Abeta peptide.

By altering key amino acid residues of the Alzheimer's disease-associated amyloid-beta peptide, we investigated the mechanism through which amyloid-beta inhibits cytochrome c oxidase (EC 1.9.3.1). Native amyloid-beta inhibited cytochrome oxidase by up to 65%, and the level of inhibition was determined by the period of amyloid-beta ageing before the cytochrome oxidase assay. Substituting tyrosine-10 with alanine did not affect maximal enzyme inhibition, but the altered peptide required a longer period of ageing. By contrast, oxidizing the sulfur of methionine-35 to a sulfoxide, or substituting methionine-35 with valine, completely abrogated the peptide's inhibitory potential towards cytochrome oxidase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that the loss of inhibitory potential towards cytochrome oxidase with the methionine-35-altered peptides did not correlate with a substantially different distribution of amyloid-beta oligomeric species. Although the amyloid-beta-mediated inhibition of cytochrome oxidase was completely dependent on the presence of divalent Cu2+, it was not supported by monovalent Cu+, and experiments with catalase and H2O2 indicated that the mechanism of cytochrome oxidase inhibition does not involve amyloid-beta-mediated H2O2 production. We propose that amyloid-beta-mediated inhibition of cytochrome oxidase is dependent on the peptide's capacity to bind, then reduce Cu2+, and that it may involve the formation of a redox active amyloid-beta-methionine radical.