Synthesis and structural characterization of a mimetic membrane-anchored prion protein

During pathogenesis of transmissible spongiform encephalopathies (TSEs) an abnormal form (PrPSc) of the host encoded prion protein (PrPC) accumulates in insoluble fibrils and plaques. The two forms of PrP appear to have identical covalent structures, but differ in secondary and tertiary structure. Both PrPC and PrPSc have glycosylphospatidylinositol (GPI) anchors through which the protein is tethered to cell membranes. Membrane attachment has been suggested to play a role in the conversion of PrPC to PrPSc, but the majority of in vitro studies of the function, structure, folding and stability of PrP use recombinant protein lacking the GPI anchor. In order to study the effects of membranes on the structure of PrP, we synthesized a GPI anchor mimetic (GPIm), which we have covalently coupled to a genetically engineered cysteine residue at the C-terminus of recombinant PrP. The lipid anchor places the protein at the same distance from the membrane as does the naturally occurring GPI anchor. We demonstrate that PrP coupled to GPIm (PrP-GPIm) inserts into model lipid membranes and that structural information can be obtained from this membrane-anchored PrP. We show that the structure of PrP-GPIm reconstituted in phosphatidylcholine and raft membranes resembles that of PrP, without a GPI anchor, in solution. The results provide experimental evidence in support of previous suggestions that NMR structures of soluble, anchor-free forms of PrP represent the structure of cellular, membrane-anchored PrP. The availability of a lipid-anchored construct of PrP provides a unique model to investigate the effects of different lipid environments on the structure and conversion mechanisms of PrP

Neutral processes dominate microbial community assembly in Atlantic salmon, Salmo salar

In recent years a wealth of studies have examined the relationships between a host and its microbiome across diverse taxa. Many studies characterise the host microbiome without considering the ecological processes that underpin microbiome assembly. In this study, the intestinal microbiota of Atlantic salmon, Salmo salar, sampled from farmed and wild environments was first characterised using 16s rDNA MiSeq sequencing analysis. We used neutral community models to determine the balance of stochastic and deterministic processes that underpin microbial community assembly and transfer across lifecycle stage and between gut compartments. Across gut compartments in farmed fish, neutral models suggest that most microbes are transient with no evidence of adaptation to their environment. In wild fish, we find declining taxonomic and functional microbial community richness as fish mature through different lifecycle stages. Alongside neutral community models applied to wild fish, we suggest declining richness demonstrates an increasing role for the host in filtering microbial communities that is correlated with age. We find a limited subset of gut microflora adapted to the farmed and wild host environment among which Mycoplasma sp. are prominent. Our study reveals the ecological drivers underpinning community assembly in both farmed and wild Atlantic salmon and underlines the importance of understanding the role of stochastic processes such as random drift and small migration rates in microbial community assembly, before considering any functional role of the gut microbes encountered

The value of diastolic function parameters in the prediction of left atrial appendage thrombus in patients with nonvalvular atrial fibrillation

BACKGROUND: Left ventricular diastolic impairment and consequently elevated filling pressure may contribute to stasis leading to left atrial appendage thrombus (LAAT) in nonvalvular atrial fibrillation (AF). We investigated whether transthoracic echocardiographic parameters can predict LAAT independent of traditional clinical predictors. METHODS: We conducted a retrospective cohort study of 297 consecutive nonvalvular AF patients who underwent transthoracic echocardiogram followed by a transesophageal echocardiogram within one year. Multivariate logistic regression analysis models were used to determine factors independently associated with LAAT. RESULTS: Nineteen subjects (6.4%) were demonstrated to have LAAT by transesophageal echocardiography. These patients had higher mean CHADS(2) scores [2.6 ± 1.2 vs. 1.9 ± 1.3, P = 0.009], higher E:e’ ratios [16.6 ± 6.1 vs. 12.0 ± 5.4, P = 0.001], and lower mean e’ velocities [6.5 ± 2.1 cm/sec vs. 9.1 ± 3.2 cm/sec, P = 0.001]. Both E:e’ and e’ velocity were associated with LAAT formation independent of the CHADS(2) score, warfarin therapy, left ventricular ejection fraction (LVEF), and left atrial volume index (LAVI) [E:e’ odds-ratio = 1.14 (95% confidence interval = 1.03 – 1.3), P = 0.009; e’ velocity odds-ratio = 0.68 (95% confidence interval = 0.5 – 0.9), P = 0.007]. Similarly, diastolic function parameters were independently associated with spontaneous echo contrast. CONCLUSION: The diastolic function indices E:e’ and e’ velocity are independently associated with LAAT in nonvalvular AF patients and may help identify patients at risk for LAAT

The Pyridoxal 5 '-Phosphate (PLP)-Dependent Enzyme Serine Palmitoyltransferase (SPT):Effects of the Small Subunits and Insights from Bacterial Mimics of Human hLCB2a HSAN1 Mutations

The pyridoxal 5′-phosphate (PLP)-dependent enzyme serine palmitoyltransferase (SPT) catalyses the first step of de novo sphingolipid biosynthesis. The core human enzyme is a membrane-bound heterodimer composed of two subunits (hLCB1 and hLCB2a/b), and mutations in both hLCB1 (e.g., C133W and C133Y) and hLCB2a (e.g., V359M, G382V, and I504F) have been identified in patients with hereditary sensory and autonomic neuropathy type I (HSAN1), an inherited disorder that affects sensory and autonomic neurons. These mutations result in substrate promiscuity, leading to formation of neurotoxic deoxysphingolipids found in affected individuals. Here we measure the activities of the hLCB2a mutants in the presence of ssSPTa and ssSPTb and find that all decrease enzyme activity. High resolution structural data of the homodimeric SPT enzyme from the bacterium Sphingomonas paucimobilis (Sp SPT) provides a model to understand the impact of the hLCB2a mutations on the mechanism of SPT. The three human hLCB2a HSAN1 mutations map onto Sp SPT (V246M, G268V, and G385F), and these mutant mimics reveal that the amino acid changes have varying impacts; they perturb the PLP cofactor binding, reduce the affinity for both substrates, decrease the enzyme activity, and, in the most severe case, cause the protein to be expressed in an insoluble form

The pharmacological regulation of cellular mitophagy

Small molecules are pharmacological tools of considerable value for dissecting complex biological processes and identifying potential therapeutic interventions. Recently, the cellular quality-control process of mitophagy has attracted considerable research interest; however, the limited availability of suitable chemical probes has restricted our understanding of the molecular mechanisms involved. Current approaches to initiate mitophagy include acute dissipation of the mitochondrial membrane potential (ΔΨm) by mitochondrial uncouplers (for example, FCCP/CCCP) and the use of antimycin A and oligomycin to impair respiration. Both approaches impair mitochondrial homeostasis and therefore limit the scope for dissection of subtle, bioenergy-related regulatory phenomena. Recently, novel mitophagy activators acting independently of the respiration collapse have been reported, offering new opportunities to understand the process and potential for therapeutic exploitation. We have summarized the current status of mitophagy modulators and analyzed the available chemical tools, commenting on their advantages, limitations and current applications

Idiopathic isolated clitoromegaly: A report of two cases

BACKGROUND: Clitoromegaly is a frequent congenital malformation, but acquired clitoral enlargement is relatively rare. METHODS: Two acquired clitoromegaly cases treated in Atatürk Training Hospital, Izmir, Turkey are presented. RESULTS: History from both patients revealed clitoromegaly over the last three years. Neither gynecological nor systemic abnormalities were detected in either patient. Karyotype analyses and hormonal tests were normal. Abdominal and gynaecological ultrasound did not show any cystic lesion or other abnormal finding. Computerized tomography scan of the adrenal glands was normal. Clitoroplasty with preservation of neurovascular pedicles was performed for the treatment of clitoromegaly. CONCLUSION: The patients were diagnosed as "idiopathic isolated" clitoromegaly. To the best of our knowledge, there has been no detailed report about idiopathic clitoromegaly in the literature

Direct Observation of Defects and Increased Ion Permeability of a Membrane Induced by Structurally Disordered Cu/Zn-Superoxide Dismutase Aggregates

Interactions between protein aggregates and a cellular membrane have been strongly implicated in many protein conformational diseases. However, such interactions for the case of Cu/Zn superoxide dismutase (SOD1) protein, which is related to fatal neurodegenerative disorder amyotrophic lateral sclerosis (ALS), have not been explored yet. For the first time, we report the direct observation of defect formation and increased ion permeability of a membrane induced by SOD1 aggregates using a supported lipid bilayer and membrane patches of human embryonic kidney cells as model membranes. We observed that aggregated SOD1 significantly induced the formation of defects within lipid membranes and caused the perturbation of membrane permeability, based on surface plasmon resonance spectroscopy, atomic force microscopy and electrophysiology. In the case of apo SOD1 with an unfolded structure, we found that it bound to the lipid membrane surface and slightly perturbed membrane permeability, compared to other folded proteins (holo SOD1 and bovine serum albumin). The changes in membrane integrity and permeability were found to be strongly dependent on the type of proteins and the amount of aggregates present. We expect that the findings presented herein will advance our understanding of the pathway by which structurally disordered SOD1 aggregates exert toxicity in vivo

Apolipoprotein A-II Influences Apolipoprotein E-Linked Cardiovascular Disease Risk in Women with High Levels of HDL Cholesterol and C-Reactive Protein

Background: In a previous report by our group, high levels of apolipoprotein E (apoE) were demonstrated to be associated with risk of incident cardiovascular disease in women with high levels of C-reactive protein (CRP) in the setting of both low (designated as HR1 subjects) and high (designated as HR2 subjects) levels of high-density lipoprotein cholesterol (HDL-C). To assess whether apolipoprotein A-II (apoA-II) plays a role in apoE-associated risk in the two female groups. Methodology/Principal: Outcome event mapping, a graphical data exploratory tool; Cox proportional hazards multivariable regression; and curve-fitting modeling were used to examine apoA-II influence on apoE-associated risk focusing on HDL particles with apolipoprotein A-I (apoA-I) without apoA-II (LpA-I) and HDL particles with both apoA-I and apoA-II (LpA-I:A-II). Results of outcome mappings as a function of apoE levels and the ratio of apoA-II to apoA-I revealed within each of the two populations, a high-risk subgroup characterized in each situation by high levels of apoE and additionally: in HR1, by a low value of the apoA-II/apoA-I ratio; and in HR2, by a moderate value of the apoA-II/apoA-I ratio. Furthermore, derived estimates of LpA-I and LpA-I:A-II levels revealed for high-risk versus remaining subjects: in HR1, higher levels of LpA-I and lower levels of LpA-I:A-II; and in HR2 the reverse, lower levels of LpA-I and higher levels of LpA-I:A-II. Results of multivariable risk modeling as a function of LpA-I and LpA-I:A-II (dichotomized as highest quartile versus combined three lower quartiles) revealed association of risk only for high levels of LpA-I:A-II in the HR2 subgroup (hazard ratio 5.31, 95% CI 1.12-25.17, p = 0.036). Furthermore, high LpA-I: A-II levels interacted with high apoE levels in establishing subgroup risk. Conclusions/Significance: We conclude that apoA-II plays a significant role in apoE-associated risk of incident CVD in women with high levels of HDL-C and CRP

Structural insights into the catalysis and regulation of E3 ubiquitin ligases

Covalent attachment (conjugation) of one or more ubiquitin molecules to protein substrates governs numerous eukaryotic cellular processes, including apoptosis, cell division and immune responses. Ubiquitylation was originally associated with protein degradation, but it is now clear that ubiquitylation also mediates processes such as protein–protein interactions and cell signalling depending on the type of ubiquitin conjugation. Ubiquitin ligases (E3s) catalyse the final step of ubiquitin conjugation by transferring ubiquitin from ubiquitin-conjugating enzymes (E2s) to substrates. In humans, more than 600 E3s contribute to determining the fates of thousands of substrates; hence, E3s need to be tightly regulated to ensure accurate substrate ubiquitylation. Recent findings illustrate how E3s function on a structural level and how they coordinate with E2s and substrates to meticulously conjugate ubiquitin. Insights regarding the mechanisms of E3 regulation, including structural aspects of their autoinhibition and activation are also emerging