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

Joshi, Shubhada N.; Butler, David; Messer, Anne

doi:10.4161/mabs.21696

Cited by 64 publications

(64 citation statements)

References 30 publications

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“…Because direct fusion of GFP to the C-terminus of α-Syn results in partial truncation of the fusion protein that spontaneously form inclusions in the neuronal cytoplasm [26], a flexible (Gly 4 Ser) 4 linker [27], was inserted between GFP and Syn to allow independent folding of the two proteins. In previous studies, VH14PEST was shown to significantly decrease α-Syn~GFP levels [14]. To rule out the possibility that VH14PEST binds to epitopes that are present only in α-Syn~GFP, we tested two additional paradigms.…”

Section: Resultsmentioning

confidence: 99%

“…Efficacy is further enhanced with bifunctional constructs that have a proteasomal targeting PEST degron to enhance the degradation during the time that the complex is associated. This degron is directly fused to intrabodies that prevent misfolding of their target antigen [13, 14]. The degron used in this study is from mouse Ornithine Decarboxylase (ODC), a short-lived protein containing a C-terminal PEST degron that has been shown to heterologously reduce the half-life of GFP transcription reporters [15–17].…”

Section: Introductionmentioning

confidence: 99%

“…Fusion with the PEST motif was able to increase cytoplasmic solubility of anti-a-syn fragments (including the unprotected VH14) due to the overall negative charge, while enhancing degradation [14]. In the current study, efficacy of VH14PEST is assessed using a series of in situ models and assays, since each models some but not all aspects of a-syn pathogenesis.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2016

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

et al. 2016

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“…Similarly, a fusion protein comprising a scFv and a specific protease can further aid in the clearance of aggregation-prone proteins [155]. Finally, the use of gene therapy with intracellular scFv (intrabodies) is also being explored for the detection and clearance of intracellular α-syn aggregates [156][157][158].…”

Section: Developing New Technologies For Immunotherapymentioning

confidence: 99%

Immunotherapeutic Approaches Targeting Amyloid-β, α-Synuclein, and Tau for the Treatment of Neurodegenerative Disorders

2016

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Disease-modifying alternatives are sorely needed for the treatment of neurodegenerative disorders, a group of diseases that afflict approximately 50 million Americans annually. Immunotherapy is one of the most developed approaches in this direction. Vaccination against amyloid-β, α-synuclein, or tau has been extensively explored, specially as the discovery that these proteins may propagate cell-to-cell and be accessible to antibodies when embedded into the plasma membrane or in the extracellular space. Likewise, the use of passive immunization approaches with specific antibodies against abnormal conformations of these proteins has also yielded promising results. The clinical development of immunotherapies for Alzheimer's disease, Parkinson's disease, frontotemporal dementia, dementia with Lewy bodies, and other neurodegenerative disorders is a field in constant evolution. Results to date suggest that immunotherapy is a promising therapeutic approach for neurodegenerative diseases that progress with the accumulation and prion-like propagation of toxic protein aggregates. Here we provide an overview of the most novel and relevant immunotherapeutic advances targeting amyloid-β in Alzheimer's disease, α-synuclein in Alzheimer's disease and Parkinson's disease, and tau in Alzheimer's disease and frontotemporal dementia.

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“…Addition of the acidic PEST peptide to four anti-α-Syn intrabodies improved the solubility for all 4, while still retaining sufficient intracellular levels of intrabody-PEST protein. In addition, the highest affinity, but least soluble of the parent constructs, the NAC-specific [81] Human scFv Oligomeric α-Syn Tomlinson I and J antibody library D10 [76] Human scFv Pan-specific Human scFv phage Griffin I library VH14 [74] Human nanobody Part of hydrophobic NAC of α-Syn Human nonimmune yeast surface display library NAC32 [74] Human scFv Part of hydrophobic NAC of α-Syn Human nonimmune yeast surface display library 6E [28] Human scFv Fibrillar α-Syn Tomlinson I and J antibody library NbSyn2 [79] Camelid VHH Monomeric α-Syn Phage display ScFv = single chain Fv; VHH = small heavy-chain-only antibody fragments; NAC = nonamyloid component human heavy-chain only nanobody, VH14, acquired the capacity to degrade α-Syn~GFP [90]. Thus, this bifunctional approach to the intrabody engineering can be used successfully to target and direct the molecule of choice to the required compartment, and shows great promise of targeting the earliest stages of neurodegeneration.…”

Section: Synmentioning

confidence: 99%

Intrabodies as Neuroprotective Therapeutics

Messer

Joshi

2013

Neurotherapeutics

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The process of misfolding of proteins that can trigger a pathogenic cascade leading to neurodegenerative diseases largely originates intracellularly. It is possible to harness the specificity and affinity of antibodies to counteract either protein misfolding itself, or the aberrant interactions and excess stressors immediately downstream of the primary insult. This review covers the emerging field of engineering intracellular antibody fragments, intrabodies and nanobodies, in neurodegeneration. Huntington's disease has provided the clearest proof of concept for this approach. The model systems and readouts for this disorder power the studies, and the potential to intervene therapeutically at early stages in known carriers with projected ages of onset increases the chances of meaningful clinical trials. Both single-chain Fv and single-domain nanobodies have been identified against specific targets; data have allowed feedback for rational design of bifunctional constructs, as well as target validation. Intrabodies that can modulate the primary accumulating protein in Parkinson's disease, alphasynuclein, are also reviewed, covering a range of domains and conformers. Recombinant antibody technology has become a major player in the therapeutic pipeline for cancer, infectious diseases, and autoimmunity. There is also tremendous potential for applying this powerful biotechnology to neurological diseases.Keywords Intrabodies . Huntington's disease . Immunotherapy . Intrabody-PEST fusions . α-synuclein . Parkinson's disease . Polyglutamine Biological Antibody Therapies for Misfolding ProteinsThe problem of misfolding proteins appears to underlie a significant fraction of neurodegenerative diseases. Protein homeostasis, often referred to as proteostasis, is an especially critical process in postmitotic neurons. The balance of pathways for protein folding, interactions, intracellular localizations, and degradation is a complex one, and can be dysregulated by combinations of mutations, environmental stressors, and aging. We have taken the neurotherapeutic approach of normalizing or manipulating proteostasis using engineered intracellular antibody fragments-intrabodies-that bind with high specificity to selected targets. These intrabodies can be selected and manipulated as genes, allowing the full spectrum of genetic engineering to produce multifunctional constructs that can alter the folding, interactions, intracellular localization, and turnover kinetics/ levels of the target protein. Intrabodies are also valuable molecular tools, which can be used to dissect the functional and pathogenic motifs in these proteins and that could be useful for the development of alternative therapeutics. In this review, we will cover the development of this approach for Huntington's disease (HD), which has a well-described target, evaluating effects in several cell culture and in vivo systems with strong readouts for therapeutic efficacy, and advances in engineering anti-HD intrabodies. We also cover a small, but growing, literature...

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

Cited by 64 publications

References 30 publications

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

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

Immunotherapeutic Approaches Targeting Amyloid-β, α-Synuclein, and Tau for the Treatment of Neurodegenerative Disorders

Intrabodies as Neuroprotective Therapeutics

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