Perturbation with Intrabodies Reveals That Calpain Cleavage Is Required for Degradation of Huntingtin Exon 1

Background: Proteolytic processing of mutant huntingtin (mHtt), the protein that causes Huntington's disease (HD), is critical for mHtt toxicity and disease progression. mHtt contains several caspase and calpain cleavage sites that generate N-terminal fragments that are more toxic than full-length mHtt. Further processing is then required for the degradation of these fragments, which in turn, reduces toxicity. This unknown, secondary degradative process represents a promising therapeutic target for HD. Methodology/Principal Findings: We have used intrabodies, intracellularly expressed antibody fragments, to gain insight into the mechanism of mutant huntingtin exon 1 (mHDx-1) clearance. Happ1, an intrabody recognizing the proline-rich region of mHDx-1, reduces the level of soluble mHDx-1 by increasing clearance. While proteasome and macroautophagy inhibitors reduce turnover of mHDx-1, Happ1 is still able to reduce mHDx-1 under these conditions, indicating Happ1-accelerated mHDx-1 clearance does not rely on these processes. In contrast, a calpain inhibitor or an inhibitor of lysosomal pH block Happ1-mediated acceleration of mHDx-1 clearance. These results suggest that mHDx-1 is cleaved by calpain, likely followed by lysosomal degradation and this process regulates the turnover rate of mHDx-1. Sequence analysis identifies amino acid (AA) 15 as a potential calpain cleavage site. Calpain cleavage of recombinant mHDx-1 in vitro yields fragments of sizes corresponding to this prediction. Moreover, when the site is blocked by binding of another intrabody, V_L12.3, turnover of soluble mHDx-1 in living cells is blocked. Conclusions/Significance: These results indicate that calpain-mediated removal of the 15 N-terminal AAs is required for the degradation of mHDx-1, a finding that may have therapeutic implications

Examination of ataxin-3 (atx-3) aggregation by structural mass spectrometry techniques: A rationale for expedited aggregation upon polyglutamine (polyQ) expansion

Expansion of polyglutamine stretches leads to the formation of polyglutamine-containing neuronal aggregates and neuronal death in nine diseases for which there currently are no treatments or cures. This is largely due to a lack in understanding of the mechanisms by which expanded polyglutamine regions contribute to aggregation and disease. To complicate matters further, several of the polyglutamine-disease related proteins, including ataxin-3, have a multistage aggregation mechanism in which flanking domain self-assembly precedes polyglutamine aggregation yet is influenced by polyglutamine expansion. How polyglutamine expansion influences flanking domain aggregation is poorly understood. Here, we use a combination of mass spectrometry and biophysical approaches to investigate this issue for ataxin-3. We show that the conformational dynamics of the flanking Josephin domain in ataxin-3 with an expanded polyglutamine tract are altered in comparison to those exhibited by its nonexpanded counterpart, specifically within the aggregation-prone region of the Josephin domain (amino acid residues 73-96). Expansion thus exposes this region more frequently in ataxin-3 containing an expanded polyglutamine tract, providing a molecular explanation of why aggregation is accelerated upon polyglutamine expansion. Here, harnessing the power of ion mobility spectrometry-mass spectrometry, oligomeric species formed during aggregation are characterized and a model for oligomer growth proposed. The results suggest that a conformational change occurs at the dimer level that initiates self-assembly. New insights into ataxin-3 fibril architecture are also described, revealing the region of the Josephin domain involved in protofibril formation and demonstrating that polyglutamine aggregation proceeds as a distinct second step after protofibril formation without requiring structural rearrangement of the protofibril core. Overall, the results enable the effect of polyglutamine expansion on every stage of ataxin-3 self-assembly, from monomer through to fibril, to be described and a rationale for expedited aggregation upon polyglutamine expansion to be provided

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

Conformational Targeting of Fibrillar Polyglutamine Proteins in Live Cells Escalates Aggregation and Cytotoxicity

Misfolding- and aggregation-prone proteins underlying Parkinson's, Huntington's and Machado-Joseph diseases, namely alpha-synuclein, huntingtin, and ataxin-3 respectively, adopt numerous intracellular conformations during pathogenesis, including globular intermediates and insoluble amyloid-like fibrils. Such conformational diversity has complicated research into amyloid-associated intracellular dysfunction and neurodegeneration. To this end, recombinant single-chain Fv antibodies (scFvs) are compelling molecular tools that can be selected against specific protein conformations, and expressed inside cells as intrabodies, for investigative and therapeutic purposes.Using atomic force microscopy (AFM) and live-cell fluorescence microscopy, we report that a human scFv selected against the fibrillar form of alpha-synuclein targets isomorphic conformations of misfolded polyglutamine proteins. When expressed in the cytoplasm of striatal cells, this conformation-specific intrabody co-localizes with intracellular aggregates of misfolded ataxin-3 and a pathological fragment of huntingtin, and enhances the aggregation propensity of both disease-linked polyglutamine proteins. Using this intrabody as a tool for modulating the kinetics of amyloidogenesis, we show that escalating aggregate formation of a pathologic huntingtin fragment is not cytoprotective in striatal cells, but rather heightens oxidative stress and cell death as detected by flow cytometry. Instead, cellular protection is achieved by suppressing aggregation using a previously described intrabody that binds to the amyloidogenic N-terminus of huntingtin. Analogous cytotoxic results are observed following conformational targeting of normal or polyglutamine-expanded human ataxin-3, which partially aggregate through non-polyglutamine domains.These findings validate that the rate of aggregation modulates polyglutamine-mediated intracellular dysfunction, and caution that molecules designed to specifically hasten aggregation may be detrimental as therapies for polyglutamine disorders. Moreover, our findings introduce a novel antibody-based tool that, as a consequence of its general specificity for fibrillar conformations and its ability to function intracellularly, offers broad research potential for a variety of human amyloid diseases

Identification of a novel site of interaction between ataxin-3 and the amyloid aggregation inhibitor polyglutamine binding peptide 1

Amyloid diseases represent a growing social and economic burden in the developed world. Understanding the assembly pathway and the inhibition of amyloid formation is key to developing therapies to treat these diseases. The neurodegenerative condition Machado–Joseph disease is characterised by the self-aggregation of the protein ataxin-3. Ataxin-3 consists of a globular N-terminal Josephin domain, which can aggregate into curvilinear protofibrils, and an unstructured, dynamically disordered C-terminal domain containing three ubiquitin interacting motifs separated by a polyglutamine stretch. Upon expansion of the polyglutamine region above 50 residues, ataxin-3 undergoes a second stage of aggregation in which long, straight amyloid fibrils form. A peptide inhibitor of polyglutamine aggregation, known as polyQ binding peptide 1, has been shown previously to prevent the maturation of ataxin-3 fibrils. However, the mechanism of this inhibition remains unclear. Using nanoelectrospray ionisation-mass spectrometry, we demonstrate that polyQ binding peptide 1 binds to monomeric ataxin-3. By investigating the ability of polyQ binding peptide 1 to bind to truncated ataxin-3 constructs lacking one or more domains, we localise the site of this interaction to a 39-residue sequence immediately C-terminal to the Josephin domain. The results suggest a new mechanism for the inhibition of polyglutamine aggregation by polyQ binding peptide 1 in which binding to a region outside of the polyglutamine tract can prevent fibril formation, highlighting the importance of polyglutamine flanking regions in controlling aggregation and disease

Malaria Rapid Diagnostic Tests: Literary Review and Recommendation for a Quality Assurance, Quality Control Algorithm.

Malaria rapid diagnostic tests (RDTs) have had an enormous global impact which contributed to the World Health Organization paradigm shift from empiric treatment to obtaining a parasitological diagnosis prior to treatment. Microscopy, the classic standard, requires significant expertise, equipment, electricity, and reagents. Alternatively, RDT\u27s lower complexity allows utilization in austere environments while achieving similar sensitivities and specificities. Worldwide, there are over 200 different RDT brands that utilize three antigens: Plasmodium histidine-rich protein 2

Heterogeneity of a Campylobacter jejuni Protein That Is Secreted through the Flagellar Filament▿

Cj0859c, or FspA, is a small, acidic protein of Campylobacter jejuni that is expressed by a σ28 promoter. Analysis of the fspA gene in 41 isolates of C. jejuni revealed two overall variants of the predicted protein, FspA1 and FspA2. Secretion of FspA occurs in broth-grown bacteria and requires a minimum flagellar structure. The addition of recombinant FspA2, but not FspA1, to INT407 cells in vitro resulted in a rapid induction of apoptosis. These data define a novel C. jejuni virulence factor, and the observed heterogeneity among fspA alleles suggests alternate virulence potential among different strains

SUMO Modification of Huntingtin and Huntington's Disease Pathology

Huntington's disease (HD) is characterized by the accumulation of a pathogenic protein, Huntingtin (Htt), that contains an abnormal polyglutamine expansion. Here, we report that a pathogenic fragment of Htt (Httex1p) can be modified either by small ubiquitin-like modifier (SUMO)-1 or by ubiquitin on identical lysine residues. In cultured cells, SUMOylation stabilizes Httex1p, reduces its ability to form aggregates, and promotes its capacity to repress transcription. In a Drosophila model of HD, SUMOylation of Httex1p exacerbates neurodegeneration, whereas ubiquitination of Httex1p abrogates neurodegeneration. Lysine mutations that prevent both SUMOylation and ubiquitination of Httex1p reduce HD pathology, indicating that the contribution of SUMOylation to HD pathology extends beyond preventing Htt ubiquitination and degradation