Atomic force microscopy differentiates discrete size distributions between membrane protein containing and empty nanolipoprotein particles

AbstractTo better understand the incorporation of membrane proteins into discoidal nanolipoprotein particles (NLPs) we have used atomic force microscopy (AFM) to image and analyze NLPs assembled in the presence of bacteriorhodopsin (bR), lipoprotein E4 n-terminal 22k fragment scaffold and DMPC lipid. The self-assembly process produced two distinct NLP populations: those containing inserted bR (bR-NLPs) and those that did not (empty-NLPs). The bR-NLPs were distinguishable from empty-NLPs by an average increase in height of 1.0 nm as measured by AFM. Streptavidin binding to biotinylated bR confirmed that the original 1.0 nm height increase corresponds to br-NLP incorporation. AFM and ion mobility spectrometry (IMS) measurements suggest that NLP size did not vary around a single mean but instead there were several subpopulations, which were separated by discrete diameters. Interestingly, when bR was present during assembly the diameter distribution was shifted to larger particles and the larger particles had a greater likelihood of containing bR than smaller particles, suggesting that membrane proteins alter the mechanism of NLP assembly

Characterization and Reconstruction of Nanolipoprotein Particles (Nlps) by Cryo-EM and Image Reconstruction

Nanolipoprotein particles (NLPs) are small 10-20 nm diameter assemblies of apolipoproteins and lipids. At Lawrence Livermore National Laboratory (LLNL), they have constructed multiple variants of these assemblies. NLPs have been generated from a variety of lipoproteins, including apolipoprotein Al, apolipophorin III, apolipoprotein E4 22K, and MSP1T2 (nanodisc, Inc.). Lipids used included DMPC (bulk of the bilayer material), DMPE (in various amounts), and DPPC. NLPs were made in either the absence or presence of the detergent cholate. They have collected electron microscopy data as a part of the characterization component of this research. Although purified by size exclusion chromatography (SEC), samples are somewhat heterogeneous when analyzed at the nanoscale by negative stained cryo-EM. Images reveal a broad range of shape heterogeneity, suggesting variability in conformational flexibility, in fact, modeling studies point to dynamics of inter-helical loop regions within apolipoproteins as being a possible source for observed variation in NLP size. Initial attempts at three-dimensional reconstructions have proven to be challenging due to this size and shape disparity. They are pursuing a strategy of computational size exclusion to group particles into subpopulations based on average particle diameter. They show here results from their ongoing efforts at statistically and computationally subdividing NLP populations to realize greater homogeneity and then generate 3D reconstructions

HCC risk stratification after cure of hepatitis C in patients with compensated advanced chronic liver disease

Background&Aims: Hepatocellular carcinoma (HCC) is a main cause of morbidity and mortality in patients with advanced chronic liver disease (ACLD) due to chronic hepatitis C and who have achieved sustained virologic response (SVR). We elaborated risk stratification algorithms for de-novo-HCC-development after SVR and validated them in an independent cohort. Methods: Derivation cohort: 527 patients with pre-treatment ACLD and SVR to interferon-free therapy were evaluated for de-novo-HCC-development. Among others, alpha-fetoprotein (AFP) and non-invasive surrogates of portal hypertension including liver stiffness measurement (LSM) were assessed pre-/post-treatment. Validation cohort: 1500 patients with compensated ACLD (cACLD) from other European centers. Results: During a median follow-up (FU) of 41 months, 22/475 cACLD (4.6%) (1.45/100patient-years)vs.12/52 decompensated patients (23.1%, 7.00/100patient-years, p<0.001) developed de-novo-HCC. Since decompensated patients were at substantial HCC-risk, we focused on cACLD for all further analyses. In cACLD, post-treatment-values showed a higher discriminative ability for patients with/without de-novo-HCC-development during FU than pre-treatment-values or absolute/relative changes. Models based on post-treatment AFP≥4.6ngxmL-1-3points, alcohol consumption (males:>30g/d/females:>20g/d)-2points (optional), age≥59year-2points, LSM≥19.0kPa-1point, and albumin<42gxL-1-1point, accurately predicted de-novo-HCC-development (bootstrapped Harrel’s C with and without considering alcohol:0.893 and 0.836). Importantly, these parameters also provided independent prognostic information in competing risk analysis and accurately stratified patients into low-(0-3points; ≈2/3 of patients) and high-risk (≥4points; ≈1/3) groups in the derivation (algorithm with alcohol consumption; 4-year HCC-risk:0%vs.16.5%) and validation (3.3%/17.5%) cohorts. An alternative approach based on age/alcohol (optional)/FU-LSM/FU-albumin (i.e., without FU-AFP) also showed a robust performance. Conclusions: Simple algorithms based on post-treatment age/albumin/LSM, and optionally, AFP and alcohol, accurately stratified de-novo-HCC-risk in cACLD patients with SVR. Approximately 2/3 were identified as having an HCC-risk <1%/y in both the derivation and validation cohort, thereby clearly falling below the cost-effectiveness threshold for HCC-surveillance. LAY SUMMARY: Simple algorithms based on age, alcohol consumption, results of blood tests (albumin and α-fetoprotein), as well as liver stiffness measurement after the end of hepatitis C treatment identify a large proportion (approximately 2/3) of patients with advanced but still asymptomatic liver disease who are at very low risk (<1%/year) of liver cancer development, and thus, might not need to undergo 6-monthly liver ultrasound

Isolation, Characterization, and Stability of Discretely-Sized Nanolipoprotein Particles Assembled with Apolipophorin-III

Background: Nanolipoprotein particles (NLPs) are discoidal, nanometer-sized particles comprised of self-assembled phospholipid membranes and apolipoproteins. NLPs assembled with human apolipoproteins have been used for myriad biotechnology applications, including membrane protein solubilization, drug delivery, and diagnostic imaging. To expand the repertoire of lipoproteins for these applications, insect apolipophorin-III (apoLp-III) was evaluated for the ability to form discretely-sized, homogeneous, and stable NLPs. Methodology: Four NLP populations distinct with regards to particle diameters (ranging in size from 10 nm to.25 nm) and lipid-to-apoLp-III ratios were readily isolated to high purity by size exclusion chromatography. Remodeling of the purified NLP species over time at 4uC was monitored by native gel electrophoresis, size exclusion chromatography, and atomic force microscopy. Purified 20 nm NLPs displayed no remodeling and remained stable for over 1 year. Purified NLPs with 10 nm and 15 nm diameters ultimately remodeled into 20 nm NLPs over a period of months. Intra-particle chemical cross-linking of apoLp-III stabilized NLPs of all sizes. Conclusions: ApoLp-III-based NLPs can be readily prepared, purified, characterized, and stabilized, suggesting their utilit

Altering APP Proteolysis: Increasing sAPPalpha Production by Targeting Dimerization of the APP Ectodomain

One of the events associated with Alzheimer's disease is the dysregulation of α- versus β-cleavage of the amyloid precursor protein (APP). The product of α-cleavage (sAPPα) has neuroprotective properties, while Aβ1-42 peptide, a product of β-cleavage, is neurotoxic. Dimerization of APP has been shown to influence the relative rate of α- and β- cleavage of APP. Thus finding compounds that interfere with dimerization of the APP ectodomain and increase the α-cleavage of APP could lead to the development of new therapies for Alzheimer's disease. Examining the intrinsic fluorescence of a fragment of the ectodomain of APP, which dimerizes through the E2 and Aβ-cognate domains, revealed significant changes in the fluorescence of the fragment upon binding of Aβ oligomers—which bind to dimers of the ectodomain— and Aβ fragments—which destabilize dimers of the ectodomain. This technique was extended to show that RERMS-containing peptides (APP695 328–332), disulfiram, and sulfiram also inhibit dimerization of the ectodomain fragment. This activity was confirmed with small angle x-ray scattering. Analysis of the activity of disulfiram and sulfiram in an AlphaLISA assay indicated that both compounds significantly enhance the production of sAPPα by 7W-CHO and B103 neuroblastoma cells. These observations demonstrate that there is a class of compounds that modulates the conformation of the APP ectodomain and influences the ratio of α- to β-cleavage of APP. These compounds provide a rationale for the development of a new class of therapeutics for Alzheimer's disease

Self-assembly and anti-amyloid cytotoxicity activity of amyloid beta peptide derivatives

The self-assembly of two derivatives of KLVFF, a fragment Abeta(16-20) of the amyloid beta (Abeta) peptide, is investigated and recovery of viability of neuroblastoma cells exposed to Abeta is observed at sub-stoichiometric peptide concentrations. Fluorescence assays show that NH2-KLVFF-CONH2 undergoes hydrophobic collapse and amyloid formation at the same critical aggregation concentration (cac). In contrast, NH2-K(Boc)LVFF-CONH2 undergoes hydrophobic collapse at a low concentration, followed by amyloid formation at a higher cac. These findings are supported by the beta-sheet features observed by FTIR. Electrospray ionization mass spectrometry indicates that NH2-K(Boc)LVFF-CONH2 forms a significant population of oligomeric species above the cac. Cryo-TEM, used together with SAXS to determine fibril dimensions, shows that the length and degree of twisting of peptide fibrils seem to be influenced by the net peptide charge. Grazing incidence X-ray scattering from thin peptide films shows features of beta-sheet ordering for both peptides, along with evidence for lamellar ordering of NH2-KLVFF-CONH2. This work provides a comprehensive picture of the aggregation properties of these two KLVFF derivatives and show their utility, in unaggregated form, in restoring the viability of neuroblastoma cells against Abeta-induced toxicity

Expression and Localization of Mitochondrial Ferritin mRNA in Alzheimer's Disease Cerebral Cortex

Mitochondrial ferritin (MtF) has been identified as a novel ferritin encoded by an intron-lacking gene with specific mitochondrial localization located on chromosome 5q23.1. MtF has been associated with neurodegenerative disorders such as Friedreich ataxia and restless leg syndrome. However, little information is available about MtF in Alzheimer's disease (AD). In this study, therefore, we investigated the expression and localization of MtF messenger RNA (mRNA) in the cerebral cortex of AD and control cases using real-time polymerase chain reaction (PCR) as well as in situ hybridization histochemistry. We also examined protein expression using western-blot assay. In addition, we used in vitro methods to further explore the effect of oxidative stress and β-amyloid peptide (Aβ) on MtF expression. To do this we examined MtF mRNA and protein expression changes in the human neuroblastoma cell line, IMR-32, after treatment with Aβ, H2O2, or both. The neuroprotective effect of MtF on oxidative stress induced by H2O2 was measured by MTT assay. The in situ hybridization studies revealed that MtF mRNA was detected mainly in neurons to a lesser degree in glial cells in the cerebral cortex. The staining intensity and the number of positive cells were increased in the cerebral cortex of AD patients. Real-time PCR and western-blot confirmed that MtF expression levels in the cerebral cortex were significantly higher in AD cases than that in control cases at both the mRNA and the protein level. Cell culture experiments demonstrated that the expression of both MtF mRNA and protein were increased by treatment with H2O2 or a combination of Aβ and H2O2, but not with Aβ alone. Finally, MtF expression showed a significant neuroprotective effect against H2O2-induced oxidative stress (p<0.05). The present study suggests that MtF is involved in the pathology of AD and may play a neuroprotective role against oxidative stress