TRIM16 controls assembly and degradation of protein aggregates by modulating the p62‐NRF2 axis and autophagy

Sequestration of protein aggregates in inclusion bodies and their subsequent degradation prevents proteostasis imbalance, cytotoxicity, and proteinopathies. The underlying molecular mechanisms controlling the turnover of protein aggregates are mostly uncharacterized. Herein, we show that a TRIM family protein, TRIM16, governs the process of stress‐induced biogenesis and degradation of protein aggregates. TRIM16 facilitates protein aggregate formation by positively regulating the p62‐NRF2 axis. We show that TRIM16 is an integral part of the p62‐KEAP1‐NRF2 complex and utilizes multiple mechanisms for stabilizing NRF2. Under oxidative and proteotoxic stress conditions, TRIM16 activates ubiquitin pathway genes and p62 via NRF2, leading to ubiquitination of misfolded proteins and formation of protein aggregates. We further show that TRIM16 acts as a scaffold protein and, by interacting with p62, ULK1, ATG16L1, and LC3B, facilitates autophagic degradation of protein aggregates. Thus, TRIM16 streamlines the process of stress‐induced aggregate clearance and protects cells against oxidative/proteotoxic stress‐induced toxicity in vitro and in vivo. Taken together, this work identifies a new mechanism of protein aggregate turnover, which could be relevant in protein aggregation‐associated diseases such as neurodegeneration.     

A tale of two tails: The importance of unstructured termini in the aggregation pathway of β2‐microglobulin

The identification of intermediate states for folding and aggregation is important from a fundamental standpoint and for the design of novel therapeutic strategies targeted at conformational disorders. Protein human β2‐microglobulin (HB2m) is classically associated with dialysis‐related amyloidosis, but the single point mutant D76N was recently identified as the causative agent of a hereditary systemic amyloidosis affecting visceral organs. Here, we use D76N as a model system to explore the early stage of the aggregation mechanism of HB2m by means of an integrative approach framed on molecular simulations. Discrete molecular dynamics simulations of a structured‐based model predict the existence of two intermediate states populating the folding landscape. The intermediate I₁ features an unstructured C‐terminus, while I₂, which is exclusively populated by the mutant, exhibits two unstructured termini. Docking simulations indicate that I₂ is the key species for aggregation at acidic and physiological pH contributing to rationalize the higher amyloidogenic potential of D76N relative to the wild‐type protein and the ΔN6 variant. The analysis carried out here recapitulates the importance of the DE‐loop in HB2m self‐association at a neutral pH and predicts a leading role of the C‐terminus and the adjacent G‐strand in the dimerization process under acidic conditions. The identification of aggregation hot‐spots is in line with experimental results that support the importance of Phe56, Asp59, Trp60, Phe62, Tyr63, and Tyr66 in HB2m amyloidogenesis. We further predict the involvement of new residues such as Lys94 and Trp95 in the aggregation process.     

Different ataxin-3 amyloid aggregates induce intracellular Ca2 + deregulation by different mechanisms in cerebellar granule cells

This work aims at elucidating the relation between morphological and physicochemical properties of different ataxin-3 (ATX3) aggregates and their cytotoxicity. We investigated a non-pathological ATX3 form (ATX3Q24), a pathological expanded form (ATX3Q55), and an ATX3 variant truncated at residue 291 lacking the polyQ expansion (ATX3/291Δ). Solubility, morphology and hydrophobic exposure of oligomeric aggregates were characterized. Then we monitored the changes in the intracellular Ca^2 + levels and the abnormal Ca^2 + signaling resulting from aggregate interaction with cultured rat cerebellar granule cells. ATX3Q55, ATX3/291Δ and, to a lesser extent, ATX3Q24 oligomers displayed similar morphological and physicochemical features and induced qualitatively comparable time-dependent intracellular Ca^2 + responses. However, only the pre-fibrillar aggregates of expanded ATX3 (the only variant which forms bundles of mature fibrils) triggered a characteristic Ca^2 + response at a later stage that correlated with a larger hydrophobic exposure relative to the two other variants. Cell interaction with early oligomers involved glutamatergic receptors, voltage-gated channels and monosialotetrahexosylganglioside (GM1)-rich membrane domains, whereas cell interaction with more aged ATX3Q55 pre-fibrillar aggregates resulted in membrane disassembly by a mechanism involving only GM1-rich areas. Exposure to ATX3Q55 and ATX3/291Δ aggregates resulted in cell apoptosis, while ATX3Q24 was substantially innocuous. Our findings provide insight into the mechanisms of ATX3 aggregation, aggregate cytotoxicity and calcium level modifications in exposed cerebellar cells.     

Oleuropein aglycone: A polyphenol with different targets against amyloid toxicity

Background

Many data highlight the benefits of the Mediterranean diet and its main lipid component, extra-virgin olive oil (EVOO). EVOO contains many phenolic compounds that have been found effective against several aging- and lifestyle-related diseases, including neurodegeneration. Oleuropein, a phenolic secoiroid glycoside, is the main polyphenol in the olive oil. It has been reported that the aglycone form of Oleuropein (OleA) interferes in vitro and in vivo with amyloid aggregation of a number of proteins/peptides involved in amyloid, particularly neurodegenerative, diseases avoiding the growth of toxic oligomers and displaying protection against cognitive deterioration.

S100A10‐Mediated Translocation of Annexin‐A2 to SNARE Proteins in Adrenergic Chromaffin Cells Undergoing Exocytosis

In neuroendocrine cells, annexin‐A2 is implicated as a promoter of monosialotetrahexosylganglioside (GM1)‐containing lipid microdomains that are required for calcium‐regulated exocytosis. As soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors (SNAREs) require a specific lipid environment to mediate granule docking and fusion, we investigated whether annexin‐A2‐induced lipid microdomains might be linked to the SNAREs present at the plasma membrane. Stimulation of adrenergic chromaffin cells induces the translocation of cytosolic annexin‐A2 to the plasma membrane, where it colocalizes with SNAP‐25 and S100A10. Cross‐linking experiments performed in stimulated chromaffin cells indicate that annexin‐A2 directly interacts with S100A10 to form a tetramer at the plasma membrane. Here, we demonstrate that S100A10 can interact with vesicle‐associated membrane protein 2 (VAMP2) and show that VAMP2 is present at the plasma membrane in resting adrenergic chromaffin cells. Tetanus toxin that cleaves VAMP2 solubilizes S100A10 from the plasma membrane and inhibits the translocation of annexin‐A2 to the plasma membrane. Immunogold labelling of plasma membrane sheets combined with spatial point pattern analysis confirmed that S100A10 is present in VAMP2 microdomains at the plasma membrane and that annexin‐A2 is observed close to S100A10 and to syntaxin in stimulated chromaffin cells. In addition, these results showed that the formation of phosphatidylinositol (4,5)‐bisphosphate (PIP₂) microdomains colocalized with S100A10 in the vicinity of docked granules, suggesting a functional interplay between annexin‐A2‐mediated lipid microdomains and SNAREs during exocytosis.     

Class I Major Histocompatibility Complex,the Trojan Horse for Secretion of Amyloidogenic β2-Microglobulin

To form extracellular aggregates, amyloidogenic proteins bypass the intracellular quality control, which normally targets unfolded/aggregated polypeptides. Human D76N β₂-microglobulin (β₂m) variant is the prototype of unstable and amyloidogenic protein that forms abundant extracellular fibrillar deposits. Here we focus on the role of the class I major histocompatibility complex (MHCI) in the intracellular stabilization of D76N β₂m. Using biophysical and structural approaches, we show that the MHCI containing D76N β₂m (MHCI₇₆) displays stability, dissociation patterns, and crystal structure comparable with those of the MHCI with wild type β₂m. Conversely, limited proteolysis experiments show a reduced protease susceptibility for D76N β₂m within the MHCI₇₆ as compared with the free variant, suggesting that the MHCI has a chaperone-like activity in preventing D76N β₂m degradation within the cell. Accordingly, D76N β₂m is normally assembled in the MHCI and circulates as free plasma species in a transgenic mouse model.     

Design of nonapeptide LVFFARKHH: A bifunctional agent against Cu2+‐mediated amyloid β‐protein aggregation and cytotoxicity

Dysfunctional accumulation of amyloid β‐protein (Aβ) mediated by Cu²⁺ exhibits higher neurotoxicity and accelerates the progress of Alzheimer's disease, so inhibition of Cu²⁺‐mediated Aβ aggregation and cytotoxicity has been considered as a therapeutic strategy for the disease. Herein, a nonapeptide was designed by linking HH to the C‐terminus of a peptide inhibitor of Aβ aggregation, LVFFARK (LK7). We found that the nonapeptide, LK7‐HH, possessed dual functionality, including enhanced inhibition capability on Aβ aggregation as compared to LK7, and chelating Cu²⁺ with a dissociation constant of 5.50 μM. This enabled LK7‐HH to arrest the generation of reactive oxygen species catalyzed by Cu²⁺ or Cu²⁺‐Aβ complex, and to inhibit Cu²⁺‐induced Aβ aggregation. Moreover, in contrast with the cytotoxicity of LK7 aggregates, LK7‐HH was biocompatible because HH conjugation made its aggregation behavior different from LK7. Thus, LK7‐HH efficiently suppressed Cu²⁺‐mediated Aβ aggregation and cytotoxicity. An equimolar concentration of LK7‐HH increased cell viability from 50% to 90% when treating Aβ₄₀‐Cu²⁺ complexes. The results provided insights into the roles of HH in enhancing the inhibition of Aβ and Cu²⁺‐induced Aβ aggregations, in eliminating Cu²⁺‐induced cytotoxicities by arresting generation of reactive oxygen species, and in making the peptide biocompatible. Therefore, this work would contribute to the design of potent peptide‐based inhibitors of Cu²⁺‐mediated Aβ aggregation and cytotoxicity.     

Hemoglobin regulates the migration of glioma cells along poly(ε‐caprolactone)‐aligned nanofibers

Aligned fibers have been shown to facilitate cell migration in the direction of fiber alignment while oxygen (O₂)‐carrying solutions improve the metabolism of cells in hypoxic culture. Therefore, U251 aggregate migration on poly(ε‐caprolactone) (PCL)‐aligned fibers was studied in cell culture media supplemented with the O₂ storage and transport protein hemoglobin (Hb) obtained from bovine, earthworm and human sources at concentrations ranging from 0 to 5 g/L within a cell culture incubator exposed to O₂ tensions ranging from 1 to 19% O₂. Individual cell migration was quantified using a wound healing assay. In addition, U251 cell aggregates were developed and aggregate dispersion/cell migration quantified on PCL‐aligned fibers. The results of this work show that the presence of bovine or earthworm Hb improved individual cell viability at 1% O₂, while human Hb adversely affected cell viability at increasing Hb concentrations and decreasing O₂ levels. The control data suggests that decreasing the O₂ tension in the incubator from 5 to 1% O₂ decreased aggregate dispersion on the PCL‐aligned fibers. However, the addition of bovine Hb at 5% O₂ significantly improved aggregate dispersion. At 19% O₂, Hb did not impact aggregate dispersion. Also at 1% O₂, aggregate dispersion appeared to increase in the presence of earthworm Hb, but only at the latter time points. Taken together, these results show that Hb‐based O₂ carriers can be utilized to improve O₂ availability and the migration of glioma spheroids on nanofibers. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1214–1220, 2014     

Nitrogen‐mediated effects of elevated CO2 on intra‐aggregate soil pore structure

Soil pore structure has a strong influence on water retention, and is itself influenced by plant and microbial dynamics such as root proliferation and microbial exudation. Although increased nitrogen (N) availability and elevated atmospheric CO₂ concentrations (eCO₂) often have interacting effects on root and microbial dynamics, it is unclear whether these biotic effects can translate into altered soil pore structure and water retention. This study was based on a long‐term experiment (7 yr at the time of sampling) in which a C₄ pasture grass (Paspalum notatum) was grown on a sandy loam soil while provided factorial additions of N and CO₂. Through an analysis of soil aggregate fractal properties supported by 3D microtomographic imagery, we found that N fertilization induced an increase in intra‐aggregate porosity and a simultaneous shift toward greater accumulation of pore space in larger aggregates. These effects were enhanced by eCO₂ and yielded an increase in water retention at pressure potentials near the wilting point of plants. However, eCO₂ alone induced changes in the opposite direction, with larger aggregates containing less pore space than under control conditions, and water retention decreasing accordingly. Results on biotic factors further suggested that organic matter gains or losses induced the observed structural changes. Based on our results, we postulate that the pore structure of many mineral soils could undergo N‐dependent changes as atmospheric CO₂ concentrations rise, having global‐scale implications for water balance, carbon storage, and related rhizosphere functions.     

d-Ribose glycates β2-microglobulin to form aggregates with high cytotoxicity through a ROS-mediated pathway

β₂-Microglobulin (β₂M) modified with advanced glycation end products (AGEs) is a major component of the amyloid deposits in hemodialysis-associated amyloidosis (HAA). However, the effect of glycation on the misfolding and aggregation of β₂M has not been studied so far. Here we examine the molecular mechanism of aggregate formation of HAA-related ribosylated β₂M in vitro. We find that the glycating agent d-ribose interacts with human β₂M to generate AGEs that form aggregates in a time-dependent manner. Ribosylated β₂M molecules are highly oligomerized compared with unglycated β₂M, and have granular morphology. Furthermore, such ribosylated β₂M aggregates show significant cytotoxicity to both human SH-SY5Y neuroblastoma and human foreskin fibroblast FS2 cells and induce intracellular reactive oxygen species (ROS). Presence of the antioxidant N-acetylcysteine (1.0 mM) attenuated intracellular ROS and prevented cell death induction in both SH-SY5Y and FS2 cells, indicating that the cytotoxicity of ribosylated β₂M aggregates depends on a ROS-mediated pathway in both cell lines. In other words, d-ribose reacts with β₂M and induces the ribosylated protein to form granular aggregates with high cytotoxicity through a ROS-mediated pathway. These findings suggest that ribosylated β₂M aggregates could contribute to the dysfunction and death of cells and could play an important role in the pathogenesis of β₂M-associated diseases such as HAA.     

G‐protein coupled receptor array technologies: Site directed immobilisation of liposomes containing the H1‐histamine or M2‐muscarinic receptors

This paper describes a novel strategy to create a microarray of G‐protein coupled receptors (GPCRs), an important group of membrane proteins both physiologically and pharmacologically. The H₁‐histamine receptor and the M₂‐muscarinic receptor were both used as model GPCRs in this study. The receptor proteins were embedded in liposomes created from the cellular membrane extracts of Spodoptera frugiperda (Sf9) insect cell culture line with its accompanying baculovirus protein insert used for overexpression of the receptors. Once captured onto a surface these liposomes provide a favourable lipidic environment for the integral membrane proteins. Site directed immobilisation of these liposomes was achieved by introduction of cholesterol‐modified oligonucleotides (oligos). These oligo/cholesterol conjugates incorporate within the lipid bilayer and were captured by the complementary oligo strand exposed on the surface. Sequence specific immobilisation was demonstrated using a quartz crystal microbalance with dissipation (QCM‐D). Confirmatory results were also obtained by monitoring fluorescent ligand binding to GPCRs captured on a spotted oligo microarray using Confocal Laser Scanning Microscopy and the ZeptoREADER microarray imaging system. Sequence specific immobilisation of such biologically important membrane proteins could lead to the development of a heterogeneous self‐sorting liposome array of GPCRs which would underpin a variety of future novel applications.     

Phospholipase A2 activity of β‐bungarotoxin is essential for induction of cytotoxicity on cerebellar granule neurons

The aim of this study was to investigate the mechanism of the cytotoxic effect of β‐bungarotoxin (β‐BuTX), a presynaptic neurotoxin, on rat cerebellar granule neurons (CGNs). The maturation of CGNs is characterized by the prominent dense neurite networks that became fragmented after treatment with β‐BuTX, and this cytotoxic effect of β‐BuTX on CGNs was in a dose‐ and time‐dependant manner. The cytotoxic effect of β‐BuTX was found to be more potent than other toxins, such as α‐BuTX, cardiotoxin, melittin, and Naja naja atra venom phospholipase A₂. Meanwhile, undifferentiated neuroblastoma neuronal cell lines, IMR‐32 and SK‐N‐MC, and astrocytes were found to be resistant to β‐BuTX. These results indicated that only the mature CGNs were sensitive to β‐BuTX insults. None of the following chemicals: antioxidants, K⁺‐channel activator, K⁺‐channel antagonists, intracellular Ca²⁺ chelator, Ca²⁺‐channel blockers, NMDA receptor antagonists, and nitric oxide synthase inhibitor tested, were able to reduce β‐BuTX‐induced cytotoxicity. However, secretory type phospholipase A₂ inhibitors (glycyrrhizin and aristolochic acid) and a free radical scavenger (5,5‐dimethyl pyrroline N‐oxide, DMPO) could attenuate not only β‐BuTX‐induced cytotoxicity but also ROS production and caspase‐3 activation. These data suggest that phospholipase A₂ activity of β‐BuTX may be responsible for free radical generation and caspase‐3 activation that accounts for the observed cytotoxic effect. It is proposed that the CGNs can be a useful tool for studying interactions of the molecules on neuronal plasma membrane with β‐BuTX that mediates the specific cytotoxicity. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2005     

Dynamics of β-Catenin Interactions with APC Protein Regulate Epithelial Tubulogenesis

Epithelial tubulogenesis involves complex cell rearrangements that require control of both cell adhesion and migration, but the molecular mechanisms regulating these processes during tubule development are not well understood. Interactions of the cytoplasmic protein, β-catenin, with several molecular partners have been shown to be important for cell signaling and cell–cell adhesion. To examine if β-catenin has a role in tubulogenesis, we tested the effect of expressing NH₂-terminal deleted β-catenins in an MDCK epithelial cell model for tubulogenesis. After one day of treatment, hepatocyte growth factor/scatter factor (HGF/ SF)-stimulated MDCK cysts initiated tubulogenesis by forming many long cell extensions. Expression of NH₂-terminal deleted β-catenins inhibited formation of these cell extensions. Both ΔN90 β-catenin, which binds to α-catenin, and ΔN131 β-catenin, which does not bind to α-catenin, inhibited formation of cell extensions and tubule development, indicating that a function of β-catenin distinct from its role in cadherin-mediated cell–cell adhesion is important for tubulogenesis. In cell extensions from parental cysts, adenomatous polyposis coli (APC) protein was localized in linear arrays and in punctate clusters at the tips of extensions. Inhibition of cell extension formation correlated with the colocalization and accumulation of NH₂-terminal deleted β-catenin in APC protein clusters and the absence of linear arrays of APC protein. Continued HGF/ SF treatment of parental cell MDCK cysts resulted in cell proliferation and reorganization of cell extensions into multicellular tubules. Similar HGF/SF treatment of cysts derived from cells expressing NH₂-terminal deleted β-catenins resulted in cells that proliferated but formed cell aggregates (polyps) within the cyst rather than tubules. Our results demonstrate an unexpected role for β-catenin in cell migration and indicate that dynamic β-catenin–APC protein interactions are critical for regulating cell migration during epithelial tubulogenesis.     

Low levels of methyl β‐cyclodextrin disrupt GluA1‐dependent synaptic potentiation but not synaptic depression

Methyl‐β‐cyclodextrin (MβCD) is a reagent that depletes cholesterol and disrupts lipid rafts, a type of cholesterol‐enriched cell membrane microdomain. Lipid rafts are essential for neuronal functions such as synaptic transmission and plasticity, which are sensitive to even low doses of MβCD. However, how MβCD changes synaptic function, such as N‐methyl‐d ‐aspartate receptor (NMDA‐R) activity, remains unclear. We monitored changes in synaptic transmission and plasticity after disrupting lipid rafts with MβCD. At low concentrations (0.5 mg/mL), MβCD decreased basal synaptic transmission and miniature excitatory post‐synaptic current without changing NMDA‐R‐mediated synaptic transmission and the paired‐pulse facilitation ratio. Interestingly, low doses of MβCD failed to deplete cholesterol or affect α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptor (AMPA‐R) and NMDA‐R levels, while clearly reducing GluA1 levels selectively in the synaptosomal fraction. Low doses of MβCD decreased the inhibitory effects of NASPM, an inhibitor for GluA2‐lacking AMPA‐R. MβCD successfully decreased NMDA‐R‐mediated long‐term potentiation but did not affect the formation of either NMDA‐R‐mediated or group I metabotropic glutamate receptor‐dependent long‐term depression. MβCD inhibited de‐depression without affecting de‐potentiation. These results suggest that MβCD regulates GluA1‐dependent synaptic potentiation but not synaptic depression in a cholesterol‐independent manner.

     

Formation of complex AChR aggregates in vitro requires α‐dystrobrevin

Efficient function at the neuromuscular junction requires high‐density aggregates of acetylcholine receptors (AChRs) to be precisely aligned with the motor nerve terminal. A collaborative effort between the motor neuron and muscle intrinsic factors drives the formation and maintenance of these AChR aggregates. α‐Dystrobrevin (αDB), a cytoplasmic protein found at the postsynaptic membrane, has been implicated in the regulation of AChR aggregate density and patterning. To investigate the contribution of αDB to the muscle intrinsic program regulating AChR aggregate development, we analyzed the formation of complex, pretzel‐like AChR aggregates on primary muscle cell cultures derived from αDB knockout (αDB‐KO) mice in the absence of nerve or agrin. In myotubes lacking αDB, complex AChR aggregates failed to form, whereas aggregates formed readily in wildtype myotubes. Five major isoforms of αDB are expressed in skeletal muscle: αDB1, αDB1(?), αDB2, αDB2(?), and αDB3. Expression of αDB1 or αDB1(?) in αDB‐KO myotubes restored formation of complex AChR aggregates similar to those in wildtype myotubes. In contrast, individual expression of αDB2, αDB2(?), αDB3, or an αDB1 phosphorylation mutant resulted in the formation of few, if any, complex AChR aggregates. Collectively, these data suggest that αDB is a significant component of the muscle intrinsic program that mediates the formation of complex AChR aggregates and that αDB's tyrosine phosphorylation sites are of particular functional importance to this program. Although the muscle intrinsic program appears to influence synaptogenesis, the formation of complex mature AChR aggregates in αDB‐KO mice (with the motor neuron present) suggests the motor neuron, not the muscle intrinsic program, is the major stimulus driving the maturation of AChRs from plaque to pretzel in vivo. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 2009     

NH2-terminal Deletion of β-Catenin Results in Stable Colocalization of Mutant β-Catenin with Adenomatous Polyposis Coli Protein and Altered MDCK Cell Adhesion

β-Catenin is essential for the function of cadherins, a family of Ca²⁺-dependent cell–cell adhesion molecules, by linking them to α-catenin and the actin cytoskeleton. β-Catenin also binds to adenomatous polyposis coli (APC) protein, a cytosolic protein that is the product of a tumor suppressor gene mutated in colorectal adenomas. We have expressed mutant β-catenins in MDCK epithelial cells to gain insights into the regulation of β-catenin distribution between cadherin and APC protein complexes and the functions of these complexes. Full-length β-catenin, β-catenin mutant proteins with NH₂-terminal deletions before (ΔN90) or after (ΔN131, ΔN151) the α-catenin binding site, or a mutant β-catenin with a COOH-terminal deletion (ΔC) were expressed in MDCK cells under the control of the tetracycline-repressible transactivator. All β-catenin mutant proteins form complexes and colocalize with E-cadherin at cell–cell contacts; ΔN90, but neither ΔN131 nor ΔN151, bind α-catenin. However, β-catenin mutant proteins containing NH₂-terminal deletions also colocalize prominently with APC protein in clusters at the tips of plasma membrane protrusions; in contrast, full-length and COOH-terminal– deleted β-catenin poorly colocalize with APC protein. NH₂-terminal deletions result in increased stability of β-catenin bound to APC protein and E-cadherin, compared with full-length β-catenin. At low density, MDCK cells expressing NH₂-terminal–deleted β-catenin mutants are dispersed, more fibroblastic in morphology, and less efficient in forming colonies than parental MDCK cells. These results show that the NH₂ terminus, but not the COOH terminus of β-catenin, regulates the dynamics of β-catenin binding to APC protein and E-cadherin. Changes in β-catenin binding to cadherin or APC protein, and the ensuing effects on cell morphology and adhesion, are independent of β-catenin binding to α-catenin. These results demonstrate that regulation of β-catenin binding to E-cadherin and APC protein is important in controlling epithelial cell adhesion.     

Lipid composition and macromolecular crowding effects on CYP2J2‐mediated drug metabolism in nanodiscs

Lipid composition and macromolecular crowding are key external effectors of protein activity and stability whose role varies between different proteins. Therefore, it is imperative to study their effects on individual protein function. CYP2J2 is a membrane‐bound cytochrome P450 in the heart involved in the metabolism of fatty acids and xenobiotics. In order to facilitate this metabolism, cytochrome P450 reductase (CPR), transfers electrons to CYP2J2 from NADPH. Herein, we use nanodiscs to show that lipid composition of the membrane bilayer affects substrate metabolism of the CYP2J2‐CPR nanodisc (ND) system. Differential effects on both NADPH oxidation and substrate metabolism by CYP2J2‐CPR are dependent on the lipid composition. For instance, sphingomyelin containing nanodiscs produced more secondary substrate metabolites than discs of other lipid compositions, implying a possible conformational change leading to processive metabolism. Furthermore, we demonstrate that macromolecular crowding plays a role in the lipid‐solubilized CYP2J2‐CPR system by increasing the K_m and decreasing the V_max, and effect that is size‐dependent. Crowding also affects the CYP2J2‐CPR‐ND system by decreasing both the K_m and V_max for Dextran‐based macromolecular crowding agents, implying an increase in substrate affinity but a lack of metabolism. Finally, protein denaturation studies show that crowding agents destabilize CYP2J2, while the multidomain protein CPR is stabilized. Overall, these studies are the first report on the role of the surrounding lipid environment and macromolecular crowding in modulating enzymatic function of CYP2J2‐CPR membrane protein system.     

Molecular design and optimization of hepatic cancer SLP76‐derived PLCγ1 SH3‐binding peptide with the systematic N‐substitution of peptide PXXP motif

The phospholipase Cγ1 (PLCγ1) is essential for T‐cell signaling and activation in hepatic cancer immune response, which has a regulatory Src homology 3 (SH3) domain that can specifically recognize and interact with the PXXP‐containing decapeptide segment (¹⁸⁵QP P VP P QRPM¹⁹⁴, termed as SLP76^185–194 peptide) of adaptor protein SLP76 following T‐cell receptor ligation. The isolated peptide can only bind to the PLCγ1 SH3 domain with a moderate affinity due to lack of protein context support. Instead of the traditional natural residue mutagenesis that is limited by low structural diversity and shifted target specificity, we herein attempt to improve the peptide affinity by replacing the two key proline residues Pro187 and Pro190 of SLP76^185–194 PXXP motif with nonnatural N‐substituted amino acids, as the proline is the only endogenous N‐substituted amino acid. The replacement would increase peptide flexibility but can restore peptide activity by establishing additional interactions with the domain. Structural analysis reveals that the domain pocket can be divided into a large amphipathic region and a small negatively charged region; they accommodate hydrophobic, aromatic, polar, and moderate‐sized N‐substituted amino acid types. A systematic replacement combination profile between the peptide residues Pro187 and Pro190 is created by structural modeling, dynamics simulation, and energetics analysis, from which six improved and two reduced N‐substituted peptides as well as native SLP76^185–194 peptide are identified and tested for their binding affinity to the recombinant protein of the human PLCγ1 SH3 domain using fluorescence‐based assays. Two N‐substituted peptides, SLP76^185–194(N‐Leu187/N‐Gln190) and SLP76^185–194(N‐Thr187/N‐Gln190), are designed to have high potency (K_d = 0.67 ± 0.18 and 1.7 ± 0.3 μM, respectively), with affinity improvement by, respectively, 8.5‐fold and 3.4‐fold relative to native peptide (K_d = 5.7 ± 1.2 μM).     

Combination of microwell structures and direct oxygenation enables efficient and size‐regulated aggregate formation of an insulin‐secreting pancreatic β‐cell line

Spherical three‐dimensional (3D) cellular aggregates are valuable for various applications such as regenerative medicine or cell‐based assays due to their stable and high functionality. However, previous methods to form aggregates have shown drawbacks, being labor‐intensive, showing low productivity per unit area or volume and difficulty to form homogeneous aggregates. We proposed a novel strategy based on oxygen‐permeable polydimethylsiloxane (PDMS) honeycomb microwell sheets, which can theoretically supply about 80 times as much oxygen as conventional polystyrene culture dishes, to produce recoverable aggregates in controllable sizes using mouse insulinoma cells (MIN6‐m9). In 48 hours of culture, the PDMS sheets produced aggregates whose diameters were strictly controlled (?32, 60, 90, 150 and 280 mm) even at an inoculum density eight times higher (8.0×105 cells/cm²) than that of normal confluent monolayers (1.0×105 cells/cm²). Measurement of the oxygen tension near the cell layer and glucose/lactate analysis clearly showed that cells exhibit aerobic respiration on the PDMS‐based culture system. Glucose‐responsive insulin secretion of the recovered aggregates showed that the aggregates around 90 mm in diameter secreted the largest amounts of insulin. This confirmed the advantages of 3D cellular organization and the existence of a suitable aggregate size, above which excess organization leads to a decreased metabolic response. These results demonstrated that this microwell‐based PDMS culture system provides a promising method to form size‐regulated and better functioning 3D cellular aggregates of various kinds of cells with a high yield per surface area. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:178–187, 2014     

Toxic HypF-N Oligomers Selectively Bind the Plasma Membrane to Impair Cell Adhesion Capability

The deposition of fibrillar protein aggregates in human organs is the hallmark of several pathological states, including highly debilitating neurodegenerative disorders and systemic amyloidoses. It is widely accepted that small oligomers arising as intermediates in the aggregation process, released by fibrils, or growing in secondary nucleation steps are the cytotoxic entities in protein-misfolding diseases, notably neurodegenerative conditions. Increasing evidence indicates that cytotoxicity is triggered by the interaction between nanosized protein aggregates and cell membranes, even though little information on the molecular details of such interaction is presently available. In this work, we propose what is, to our knowledge, a new approach, based on the use of single-cell force spectroscopy applied to multifunctional substrates, to study the interaction between protein oligomers, cell membranes, and/or the extracellular matrix. We compared the interaction of single Chinese hamster ovary cells with two types of oligomers (toxic and nontoxic) grown from the N-terminal domain of the Escherichia coli protein HypF. We were able to quantify the affinity between both oligomer type and the cell membrane by measuring the mechanical work needed to detach the cells from the aggregates, and we could discriminate the contributions of the membrane lipid and protein fractions to such affinity. The fundamental role of the ganglioside GM1 in the membrane-oligomers interaction was also highlighted. Finally, we observed that the binding of toxic oligomers to the cell membrane significantly affects the functionality of adhesion molecules such as Arg-Gly-Asp binding integrins, and that this effect requires the presence of the negatively charged sialic acid moiety of GM1.