Fusion to a highly charged proteasomal retargeting sequence increases soluble cytoplasmic expression and efficacy of diverse anti-synuclein intrabodies

Intrabodies can be powerful reagents to effect modulation of aberrant intracellular proteins that underlie a range of diseases. However, their cytoplasmic solubility can be limiting. We previously reported that overall charge and hydrophilicity can be combined to provide initial estimates of intracellular solubility, and that charge engineering via fusion can alter solubility properties experimentally. Additional studies showed that fusion of a proteasome-targeting PEST motif to the anti-huntingtin intrabody scFv-C4 can degrade mutant huntingtin proteins by directing them to the proteasome, while also increasing the negative charge. We now validate the generality of this approach with intrabodies against α-synuclein (α-syn), an important target in Parkinson disease. In this study, fusion of the PEST sequence to a set of four diverse, poorly soluble anti-α-syn intrabodies (D5E, 10H, D10 scFv, VH14 nanobody) significantly increased steady-state soluble intrabody protein levels in all cases, despite fusion with the PEST proteasomal-targeting signal. Furthermore, adding this PEST motif to the least soluble construct, VH14, significantly enhanced degradation of the target protein, α-syn~GFP. The intrabody-PEST fusion approach thus has dual advantages of potentially solubilizing intrabodies and enhancing their functionality in parallel. Empirical testing of intrabody-PEST fusions is recommended for enhancement of intrabody solubility from diverse sources.     

Bifunctional Anti-Non-Amyloid Component α-Synuclein Nanobodies Are Protective In Situ

Misfolding, abnormal accumulation, and secretion of α-Synuclein (α-Syn) are closely associated with synucleinopathies, including Parkinson’s disease (PD). VH14 is a human single domain intrabody selected against the non-amyloid component (NAC) hydrophobic interaction region of α-Syn, which is critical for initial aggregation. Using neuronal cell lines, we show that as a bifunctional nanobody fused to a proteasome targeting signal, VH14PEST can counteract heterologous proteostatic effects of mutant α-Syn on mutant huntingtin Exon1 and protect against α-Syn toxicity using propidium iodide or Annexin V readouts. We compared this anti-NAC candidate to NbSyn87, which binds to the C-terminus of α-Syn. NbSyn87PEST degrades α-Syn as well or better than VH14PEST. However, while both candidates reduced toxicity, VH14PEST appears more effective in both proteostatic stress and toxicity assays. These results show that the approach of reducing intracellular monomeric targets with novel antibody engineering technology should allow in vivo modulation of proteostatic pathologies.     

Single domain intracellular antibodies: a minimal fragment for direct in vivo selection of antigen-specific intrabodies

There is a major need in target validation and therapeutic applications for molecules that can interfere with protein function inside cells. Intracellular antibodies (intrabodies) can bind to specific targets in cells but isolation of intrabodies is currently difficult. Intrabodies are normally single chain Fv fragments comprising variable domains of the immunoglobulin heavy (VH) and light chains (VL). We now demonstrate that single VH domains have excellent intracellular properties of solubility, stability and expression within the cells of higher organisms and can exhibit specific antigen recognition in vivo. We have used this intracellular single variable domain (IDab) format, based on a previously characterised intrabody consensus scaffold, to generate diverse intrabody libraries for direct in vivo screening. IDabs were isolated using two distinct antigens and affinities of isolated IDabs ranged between 20 nM and 200 nM. Moreover, IDabs selected for binding to the RAS protein could inhibit RAS-dependent oncogenic transformation of NIH3T3 cells. The IDab format is therefore ideal for in vivo intrabody use. This approach to intrabodies obviates the need for phage antibody libraries, avoids the requirement for production of antigen in vitro and allows for direct selection of intrabodies in vivo.     

Lewy Body-like α-Synuclein Aggregates Resist Degradation and Impair Macroautophagy

Cytoplasmic α-synuclein (α-syn) aggregates, referred to as Lewy bodies, are pathological hallmarks of a number of neurodegenerative diseases, most notably Parkinson disease. Activation of macroautophagy is suggested to facilitate degradation of certain proteinaceous inclusions, but it is unclear if this pathway is capable of degrading α-syn aggregates. Here, we examined this issue by utilizing cellular models in which intracellular Lewy body-like α-syn inclusions accumulate after internalization of pre-formed α-syn fibrils into α-syn-expressing HEK293 cells or cultured primary neurons. We demonstrate that α-syn inclusions cannot be effectively degraded, even though they co-localize with essential components of both the autophagic and proteasomal protein degradation pathways. The α-syn aggregates persist even after soluble α-syn levels have been substantially reduced, suggesting that once formed, the α-syn inclusions are refractory to clearance. Importantly, we also find that α-syn aggregates impair overall macroautophagy by reducing autophagosome clearance, which may contribute to the increased cell death that is observed in aggregate-bearing cells.     

The interplay between lipids and dopamine on α-synuclein oligomerization and membrane binding

The deposition of α-syn (α-synuclein) as amyloid fibrils and the selective loss of DA (dopamine) containing neurons in the substantia nigra are two key features of PD (Parkinson''s disease). α-syn is a natively unfolded protein and adopts an α-helical conformation upon binding to lipid membrane. Oligomeric species of α-syn have been proposed to be the pathogenic species associated with PD because they can bind lipid membranes and disrupt membrane integrity. DA is readily oxidized to generate reactive intermediates and ROS (reactive oxygen species) and in the presence of DA, α-syn form of SDS-resistant soluble oligomers. It is postulated that the formation of the α-syn:DA oligomers involves the cross-linking of DA-melanin with α-syn, via covalent linkage, hydrogen and hydrophobic interactions. We investigate the effect of lipids on DA-induced α-syn oligomerization and studied the ability of α-syn:DA oligomers to interact with lipids vesicles. Our results show that the interaction of α-syn with lipids inhibits the formation of DA-induced α-syn oligomers. Moreover, the α-syn:DA oligomer cannot interact with lipid vesicles or cause membrane permeability. Thus, the formation of α-syn:DA oligomers may alter the actions of α-syn which require membrane association, leading to disruption of its normal cellular function.     

The N-Terminal Residues 43 to 60 Form the Interface for Dopamine Mediated α-Synuclein Dimerisation

α-synuclein (α-syn) is a major component of the intracellular inclusions called Lewy bodies, which are a key pathological feature in the brains of Parkinson’s disease patients. The neurotransmitter dopamine (DA) inhibits the fibrillisation of α-syn into amyloid, and promotes α-syn aggregation into SDS-stable soluble oligomers. While this inhibition of amyloid formation requires the oxidation of both DA and the methionines in α-syn, the molecular basis for these processes is still unclear. This study sought to define the protein sequences required for the generation of oligomers. We tested N- (α-syn residues 43–140) and C-terminally (1–95) truncated α-syn, and found that similar to full-length protein both truncated species formed soluble DA:α-syn oligomers, albeit 1–95 had a different profile. Using nuclear magnetic resonance (NMR), and the N-terminally truncated α-syn 43–140 protein, we analysed the structural characteristics of the DA:α-syn 43–140 dimer and α-syn 43–140 monomer and found the dimerisation interface encompassed residues 43 to 60. Narrowing the interface to this small region will help define the mechanism by which DA mediates the formation of SDS-stable soluble DA:α-syn oligomers.     

Excess Nitric Oxide Activates TRPV1-Ca2+-Calpain Signaling and Promotes PEST-dependent Degradation of Liver X Receptor α

Excess nitric oxide (NO) deregulates cholesterol metabolism in macrophage foam cells, yet the underlying molecular mechanism is incompletely understood. To investigate the mechanism, we found that in macrophages, treatment with NO donors S-nitroso-N-acetyl-D,L-penicillamine (SNAP) or diethylenetriamine/nitric oxide induced LXRα degradation and reduced the expression of the downstream target of LXRα, ATP-binding cassette transporter A1 (ABCA1), and cholesterol efflux. In addition, SNAP induced calcium (Ca²⁺) influx into cells, increased calpain activity and promoted the formation of calpain-LXRα complex. Pharmacological inhibition of calpain activity reversed the SNAP-induced degradation of LXRα, down-regulation of ABCA1 and impairment of cholesterol efflux in macrophages. SNAP increased the formation of calpain-LXRα complex in a Pro-Glu-Ser-Thr (PEST) motif-dependent manner. Truncation of the PEST motif in LXRα abolished the calpain-dependent proteolysis. Removal of extracellular Ca²⁺ by EGTA or pharmacological inhibition of TRPV1 channel activity diminished SNAP-induced increase in intracellular Ca²⁺, calpain activation, LXRα degradation, ABCA1 down-regulation and impaired cholesterol efflux. In conclusion, excess NO may activate calpain via TRPV1-Ca²⁺ signaling and promote the recognition of calpain in the PEST motif of LXRα, thereby leading to degradation of LXRα and, ultimately, downregulated ABCA1 expression and impaired ABCA1-dependent cholesterol efflux in macrophages.     

Enhanced intracellular stability of sFv-Fc fusion intrabodies

The ability of intracellular antibodies (intrabodies) to block the function of a target protein can be dependent on the stability of the single-chain antibody (sFv) when expressed in the intracellular environment. Low-affinity sFvs capable of reaching high steady-state levels can be more effective modulators of target proteins than high-affinity, unstable sFvs. In an effort to enhance the intracellular stability of sFvs when expressed as intrabodies, we have generated novel sFv-Fc fusion intrabodies. Fusion of the native sFv sequence with the entire heavy chain constant region fragment of IgG results in decreased turnover of the intrabody and enhanced steady-state accumulation of sFv-Fc protein, while maintaining the ability to target intrabody expression to sub-cellular compartments. Here, we describe the rationale and design for this strategy using two anti-cyclin E sFvs constructed for use as intrabodies.     

Bifunctional anti-huntingtin proteasome-directed intrabodies mediate efficient degradation of mutant huntingtin exon 1 protein fragments

Huntington's disease (HD) is a fatal autosomal dominant neurodegenerative disorder caused by a trinucleotide (CAG)(n) repeat expansion in the coding sequence of the huntingtin gene, and an expanded polyglutamine (>37Q) tract in the protein. This results in misfolding and accumulation of huntingtin protein (htt), formation of neuronal intranuclear and cytoplasmic inclusions, and neuronal dysfunction/degeneration. Single-chain Fv antibodies (scFvs), expressed as intrabodies that bind htt and prevent aggregation, show promise as immunotherapeutics for HD. Intrastriatal delivery of anti-N-terminal htt scFv-C4 using an adeno-associated virus vector (AAV2/1) significantly reduces the size and number of aggregates in HDR6/1 transgenic mice; however, this protective effect diminishes with age and time after injection. We therefore explored enhancing intrabody efficacy via fusions to heterologous functional domains. Proteins containing a PEST motif are often targeted for proteasomal degradation and generally have a short half life. In ST14A cells, fusion of the C-terminal PEST region of mouse ornithine decarboxylase (mODC) to scFv-C4 reduces htt exon 1 protein fragments with 72 glutamine repeats (httex1-72Q) by ~80-90% when compared to scFv-C4 alone. Proteasomal targeting was verified by either scrambling the mODC-PEST motif, or via proteasomal inhibition with epoxomicin. For these constructs, the proteasomal degradation of the scFv intrabody proteins themselves was reduced<25% by the addition of the mODC-PEST motif, with or without antigens. The remaining intrabody levels were amply sufficient to target N-terminal httex1-72Q protein fragment turnover. Critically, scFv-C4-PEST prevents aggregation and toxicity of httex1-72Q fragments at significantly lower doses than scFv-C4. Fusion of the mODC-PEST motif to intrabodies is a valuable general approach to specifically target toxic antigens to the proteasome for degradation.     

Sequential Extraction of Soluble and Insoluble Alpha-Synuclein from Parkinsonian Brains

Alpha-synuclein (α-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, oligomers and fibrils. During disease, α-syn undergoes conformational changes to form oligomers and high molecular weight aggregates that tend to make the protein more insoluble. Abnormally aggregated α-syn is a neuropathological feature of Parkinson''s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Biochemical characterization and analysis of insoluble α-syn using buffers with increasing detergent strength and high-speed ultracentrifugation provides a powerful tool to determine the development of α-syn pathology associated with disease progression. This protocol describes the isolation of increasingly insoluble/aggregated α-syn from post-mortem human brain tissue. This methodology can be adapted with modifications to studies of normal and abnormal α-syn biology in transgenic animal models harbouring different α-syn mutations as well as in other neurodegenerative diseases that feature aberrant fibrillar deposits of proteins related to their respective pathologies.     

α-synuclein assemblies sequester neuronal α3-Na+/K+-ATPase and impair Na+ gradient

Extracellular α-synuclein (α-syn) assemblies can be up-taken by neurons; however, their interaction with the plasma membrane and proteins has not been studied specifically. Here we demonstrate that α-syn assemblies form clusters within the plasma membrane of neurons. Using a proteomic-based approach, we identify the α3-subunit of Na⁺/K⁺-ATPase (NKA) as a cell surface partner of α-syn assemblies. The interaction strength depended on the state of α-syn, fibrils being the strongest, oligomers weak, and monomers none. Mutations within the neuron-specific α3-subunit are linked to rapid-onset dystonia Parkinsonism (RDP) and alternating hemiplegia of childhood (AHC). We show that freely diffusing α3-NKA are trapped within α-syn clusters resulting in α3-NKA redistribution and formation of larger nanoclusters. This creates regions within the plasma membrane with reduced local densities of α3-NKA, thereby decreasing the efficiency of Na⁺ extrusion following stimulus. Thus, interactions of α3-NKA with extracellular α-syn assemblies reduce its pumping activity as its mutations in RDP/AHC.     

Effects of Serine 129 Phosphorylation on α-Synuclein Aggregation,Membrane Association,and Internalization

Although trace levels of phosphorylated α-synuclein (α-syn) are detectable in normal brains, nearly all α-syn accumulated within Lewy bodies in Parkinson disease brains is phosphorylated on serine 129 (Ser-129). The role of the phosphoserine residue and its effects on α-syn structure, function, and intracellular accumulation are poorly understood. Here, co-expression of α-syn and polo-like kinase 2 (PLK2), a kinase that targets Ser-129, was used to generate phosphorylated α-syn for biophysical and biological characterization. Misfolding and fibril formation of phosphorylated α-syn isoforms were detected earlier, although the fibrils remained phosphatase- and protease-sensitive. Membrane binding of α-syn monomers was differentially affected by phosphorylation depending on the Parkinson disease-linked mutation. WT α-syn binding to presynaptic membranes was not affected by phosphorylation, whereas A30P α-syn binding was greatly increased, and A53T α-syn was slightly lower, implicating distal effects of the carboxyl- on amino-terminal membrane binding. Endocytic vesicle-mediated internalization of pre-formed fibrils into non-neuronal cells and dopaminergic neurons matched the efficacy of α-syn membrane binding. Finally, the disruption of internalized vesicle membranes was enhanced by the phosphorylated α-syn isoforms, a potential means for misfolded extracellular or lumenal α-syn to access cytosolic α-syn. Our results suggest that the threshold for vesicle permeabilization is evident even at low levels of α-syn internalization and are relevant to therapeutic strategies to reduce intercellular propagation of α-syn misfolding.     

Intracellular Screening of a Peptide Library to Derive a Potent Peptide Inhibitor of α-Synuclein Aggregation

Aggregation of α-synuclein (α-syn) into toxic fibrils is a pathogenic hallmark of Parkinson disease (PD). Studies have focused largely on residues 71–82, yet most early-onset mutations are located between residues 46 and 53. A semirationally designed 209,952-member library based entirely on this region was constructed, containing all wild-type residues and changes associated with early-onset PD. Intracellular cell survival screening and growth competition isolated a 10-residue peptide antagonist that potently inhibits α-syn aggregation and associated toxicity at a 1:1 stoichiometry. This was verified using continuous growth measurements and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cytotoxicity studies. Atomic force microscopy and circular dichroism on the same samples showed a random-coil structure and no oligomers. A new region of α-syn for inhibitor targeting has been highlighted, together with the approach of using a semirational design and intracellular screening. The peptides can then be used as candidates for modification in drugs capable of slowing or even preventing the onset of PD.     

Intrabodies based on intracellular capture frameworks that bind the RAS protein with high affinity and impair oncogenic transformation

We have applied in vivo intracellular antibody capture (IAC) technology to isolate human intrabodies which bind to the oncogenic RAS protein. IAC facilitates the capture of antibody fragments, in this case single-chain Fvs (scFvs), which tolerate reducing environments, such as the cytoplasm of cancer cells. Three anti-RAS scFvs with different affinity, solubility and intracellular binding activity were characterized. The anti-RAS scFvs with highest affinity were expressed relatively poorly in mammalian cells, and greater soluble expression was achieved by mutating the antibody framework to canonical consensus scaffolds, previously derived from IAC, without losing antigen specificity. Mutagenesis experiments showed that the consensus scaffolds are functional as intrabody fragments without an intra-domain disulfide bond. Furthermore, we could convert an intrabody which does not bind RAS in mammalian cells into a high-affinity reagent capable of inhibiting RAS-mediated NIH 3T3 transformation by exchanging VH and VL complementarity-determining regions onto its consensus scaffold. These data show that the consensus scaffold is a robust framework by which to improve intrabody function.     

Role of post-translational modifications on the alpha-synuclein aggregation-related pathogenesis of Parkinson’s disease

Together with neuronal loss, the existence of insoluble inclusions of alpha-synuclein (α-syn) in the brain is widely accepted as a hallmark of synucleinopathies including Parkinson’s disease (PD), multiple system atrophy, and dementia with Lewy body. Because the α-syn aggregates are deeply involved in the pathogenesis, there have been many attempts to demonstrate the mechanism of the aggregation and its potential causative factors including post-translational modifications (PTMs). Although no concrete conclusions have been made based on the previous study results, growing evidence suggests that modifications such as phosphorylation and ubiquitination can alter α-syn characteristics to have certain effects on the aggregation process in PD; either facilitating or inhibiting fibrillization. In the present work, we reviewed studies showing the significant impacts of PTMs on α-syn aggregation. Furthermore, the PTMs modulating α-syn aggregation-induced cell death have been discussed.      

DJ-1 Modulates α-Synuclein Aggregation State in a Cellular Model of Oxidative Stress: Relevance for Parkinson's Disease and Involvement of HSP70

Background

Parkinson''s disease (PD) is a neurodegenerative pathology whose molecular etiopathogenesis is not known. Novel contributions have come from familial forms of PD caused by alterations in genes with apparently unrelated physiological functions. The gene coding for alpha-synuclein (α-syn) (PARK1) has been investigated as α-syn is located in Lewy bodies (LB), intraneuronal inclusions in the substantia nigra (SN) of PD patients. A-syn has neuroprotective chaperone-like and antioxidant functions and is involved in dopamine storage and release. DJ-1 (PARK7), another family-PD-linked gene causing an autosomal recessive form of the pathology, shows antioxidant and chaperone-like activities too.

Methodology/Principal Findings

The present study addressed the question whether α-syn and DJ-1 interact functionally, with a view to finding some mechanism linking DJ-1 inactivation and α-syn aggregation and toxicity. We developed an in vitro model of α-syn toxicity in the human neuroblastoma cell line SK-N-BE, influencing DJ-1 and α-syn intracellular concentrations by exogenous addition of the fusion proteins TAT-α-syn and TAT-DJ-1; DJ-1 was inactivated by the siRNA method. On a micromolar scale TAT-α-syn aggregated and triggered neurotoxicity, while on the nanomolar scale it was neuroprotective against oxidative stress (induced by H₂O₂ or 6-hydroxydopamine). TAT-DJ-1 increased the expression of HSP70, while DJ-1 silencing made SK-N-BE cells more susceptible to oxidative challenge, rendering TAT-α-syn neurotoxic at nanomolar scale, with the appearance of TAT-α-syn aggregates.

Conclusion/Significance

DJ-1 inactivation may thus promote α-syn aggregation and the related toxicity, and in this model HSP70 is involved in the antioxidant response and in the regulation of α-syn fibril formation.     

A protein silencing switch by ligand-induced proteasome-targeting intrabodies

The selective knock-down of cellular proteins has proven useful for in vivo studies of protein function and RNAi methods are readily available for this purpose. However, interfering directly at the protein level may have distinct advantages, with the intracellular targeting of antibodies (intrabodies) representing an attractive option, although not a general one. We demonstrate a novel, general strategy named suicide (or silencing) intrabody technology (SIT), based on the inducible degradation of intrabodies, which are equipped with proteasome-targeting sequences and thus converted into suicide intrabodies. We show that suicide intrabodies are able to redirect the target cellular proteins upon stimulus administration to the proteolytic machinery, thus resulting in selective protein knock-down. Remarkably, suicide intrabody acts in a catalytic fashion. SIT is a ligand-inducible strategy, potentially applicable to any protein of interest and does not require the engineering of cellular proteolytic enzymes. SIT represents a general approach to confer “neutralizing” properties to any intrabody, a valuable feature, given the present impossibility to select a priori intrinsically neutralizing antibodies. This knock-down strategy, together with available methods to isolate functional intrabodies, should allow the large-scale investigation of intracellular protein networks.     

In-Cell Intrabody Selection from a Diverse Human Library Identifies C12orf4 Protein as a New Player in Rodent Mast Cell Degranulation

The high specificity of antibodies for their antigen allows a fine discrimination of target conformations and post-translational modifications, making antibodies the first choice tool to interrogate the proteome. We describe here an approach based on a large-scale intracellular expression and selection of antibody fragments in eukaryotic cells, so-called intrabodies, and the subsequent identification of their natural target within living cell. Starting from a phenotypic trait, this integrated system allows the identification of new therapeutic targets together with their companion inhibitory intrabody. We applied this system in a model of allergy and inflammation. We first cloned a large and highly diverse intrabody library both in a plasmid and a retroviral eukaryotic expression vector. After transfection in the RBL-2H3 rat basophilic leukemia cell line, we performed seven rounds of selection to isolate cells displaying a defect in FcεRI-induced degranulation. We used high throughput sequencing to identify intrabody sequences enriched during the course of selection. Only one intrabody was common to both plasmid and retroviral selections, and was used to capture and identify its target from cell extracts. Mass spectrometry analysis identified protein RGD1311164 (C12orf4), with no previously described function. Our data demonstrate that RGD1311164 is a cytoplasmic protein implicated in the early signaling events following FcεRI-induced cell activation. This work illustrates the strength of the intrabody-based in-cell selection, which allowed the identification of a new player in mast cell activation together with its specific inhibitor intrabody.     

A Highlights from MBoC Selection: Serine 129 phosphorylation of membrane-associated α-synuclein modulates dopamine transporter function in a G protein–coupled receptor kinase–dependent manner

Most α-synuclein (α-syn) deposited in Lewy bodies, the pathological hallmark of Parkinson disease (PD), is phosphorylated at Ser-129. However, the physiological and pathological roles of this modification are unclear. Here we investigate the effects of Ser-129 phosphorylation on dopamine (DA) uptake in dopaminergic SH-SY5Y cells expressing α-syn. Subcellular fractionation of small interfering RNA (siRNA)–treated cells shows that G protein–coupled receptor kinase 3 (GRK3), GRK5, GRK6, and casein kinase 2 (CK2) contribute to Ser-129 phosphorylation of membrane-associated α-syn, whereas cytosolic α-syn is phosphorylated exclusively by CK2. Expression of wild-type α-syn increases DA uptake, and this effect is diminished by introducing the S129A mutation into α-syn. However, wild-type and S129A α-syn equally increase the cell surface expression of dopamine transporter (DAT) in SH-SY5Y cells and nonneuronal HEK293 cells. In addition, siRNA-mediated knockdown of GRK5 or GRK6 significantly attenuates DA uptake without altering DAT cell surface expression, whereas knockdown of CK2 has no effect on uptake. Taken together, our results demonstrate that membrane-associated α-syn enhances DA uptake capacity of DAT by GRKs-mediated Ser-129 phosphorylation, suggesting that α-syn modulates intracellular DA levels with no functional redundancy in Ser-129 phosphorylation between GRKs and CK2.