Three on Three: Universal and High-Affinity Molecular Recognition of the Symmetric Homotrimeric Spike Protein of SARS-CoV-2 with a Symmetric Homotrimeric Aptamer.

Our previously discovered monomeric aptamer for SARS-CoV-2 (MSA52) possesses a universal affinity for COVID-19 spike protein variants but is ultimately limited by its ability to bind only one subunit of the spike protein. The symmetrical shape of the homotrimeric SARS-CoV-2 spike protein presents the opportunity to create a matching homotrimeric molecular recognition element that is perfectly complementary to its structural scaffold, causing enhanced binding affinity. Here, we describe a branched homotrimeric aptamer with three-fold rotational symmetry, named TMSA52, that not only possesses excellent binding affinity but is also capable of binding several SARS-CoV-2 spike protein variants with picomolar affinity, as well as pseudotyped lentiviruses expressing SARS-CoV-2 spike protein variants with femtomolar affinity. Using Pd-Ir nanocubes as nanozymes in an enzyme-linked aptamer binding assay (ELABA), TMSA52 was capable of sensitively detecting diverse pseudotyped lentiviruses in pooled human saliva with a limit of detection as low as 6.3 × 103 copies/mL. The ELABA was also used to test 50 SARS-CoV-2-positive and 60 SARS-CoV-2-negative patient saliva samples, providing sensitivity and specificity values of 84.0 and 98.3%, respectively, thus highlighting the potential of TMSA52 for the development of future rapid tests.

A DNA Barcode-Based Aptasensor Enables Rapid Testing of Porcine Epidemic Diarrhea Viruses in Swine Saliva Using Electrochemical Readout.

Pen-side testing of farm animals for infectious diseases is critical for preventing transmission in herds and providing timely intervention. However, most existing pathogen tests have to be conducted in centralized labs with sample-to-result times of 2-4 days. Herein we introduce a test that uses a dual-electrode electrochemical chip (DEE-Chip) and a barcode-releasing electroactive aptamer for rapid on-farm detection of porcine epidemic diarrhea viruses (PEDv). The sensor exploits inter-electrode spacing reduction and active field mediated transport to accelerate barcode movement from electroactive aptamers to the detection electrode, thus expediting assay operation. The test yielded a clinically relevant limit-of-detection of 6 nM (0.37 µg mL-1 ) in saliva-spiked PEDv samples. Clinical evaluation of this biosensor with 12 porcine saliva samples demonstrated a diagnostic sensitivity of 83 % and specificity of 100 % with a concordance value of 92 % at an analysis time of one hour.

A Highly Specific DNA Aptamer for RNase H2 from Clostridium difficile.

Molecular recognition elements with high specificity are of great importance for the study of molecular interactions, accurate diagnostics, drug design, and personalized medicine. Herein, a highly specific DNA aptamer for RNase H2 from Clostridium difficile (C. difficile) was generated by SELEX and minimized to 40 nucleotides. The aptamer exhibits a dissociation constant (Kd) of 1.8 ± 0.5 nM and an inhibition constant (IC50) of 7.1 ± 0.6 nM for C. difficile RNase H2, both of which are 2 orders of magnitude better for the same enzyme from other control bacteria. The fluorescent version of the aptamer can distinguish C. difficile from several other control bacteria in a cell lysate assay. This work demonstrates that a ubiquitous protein like RNase H2 can still be used as the target for the development of highly specific aptamers and the combination of the protein and the aptamer can achieve the recognition specificity needed for a diagnostic test and drug development.

High-mobility group box 1 links sensing of reactive oxygen species by huntingtin to its nuclear entry.

Huntington's disease (HD) is a neurodegenerative, age-onset disorder caused by a CAG DNA expansion in exon 1 of the HTT gene, resulting in a polyglutamine expansion in the huntingtin protein. Nuclear accumulation of mutant huntingtin is a hallmark of HD, resulting in elevated mutant huntingtin levels in cell nuclei. Huntingtin is normally retained at the endoplasmic reticulum via its N17 amphipathic α-helix domain but is released by oxidation of Met-8 during reactive oxygen species (ROS) stress. Huntingtin enters the nucleus via an importin ß1- and 2-dependent proline-tyrosine nuclear localization signal (PY-NLS), which has a unique intervening sequence in huntingtin. Here, we have identified the high-mobility group box 1 (HMGB1) protein as an interactor of the intervening sequence within the PY-NLS. Nuclear levels of HMGB1 positively correlated with varying levels of nuclear huntingtin in both HD and normal human fibroblasts. We also found that HMGB1 interacts with the huntingtin N17 region and that this interaction is enhanced by the presence of ROS and phosphorylation of critical serine residues in the N17 region. We conclude that HMGB1 is a huntingtin N17/PY-NLS ROS-dependent interactor, and this protein bridging is essential for relaying ROS sensing by huntingtin to its nuclear entry during ROS stress. ROS may therefore be a critical age-onset stress that triggers nuclear accumulation of mutant huntington in Huntington's disease.

N6-Furfuryladenine is protective in Huntington's disease models by signaling huntingtin phosphorylation.

The huntingtin N17 domain is a modulator of mutant huntingtin toxicity and is hypophosphorylated in Huntington's disease (HD). We conducted high-content analysis to find compounds that could restore N17 phosphorylation. One lead compound from this screen was N6-furfuryladenine (N6FFA). N6FFA was protective in HD model neurons, and N6FFA treatment of an HD mouse model corrects HD phenotypes and eliminates cortical mutant huntingtin inclusions. We show that N6FFA restores N17 phosphorylation levels by being salvaged to a triphosphate form by adenine phosphoribosyltransferase (APRT) and used as a phosphate donor by casein kinase 2 (CK2). N6FFA is a naturally occurring product of oxidative DNA damage. Phosphorylated huntingtin functionally redistributes and colocalizes with CK2, APRT, and N6FFA DNA adducts at sites of induced DNA damage. We present a model in which this natural product compound is salvaged to provide a triphosphate substrate to signal huntingtin phosphorylation via CK2 during low-ATP stress under conditions of DNA damage, with protective effects in HD model systems.

Huntingtin is a scaffolding protein in the ATM oxidative DNA damage response complex.

Huntington's disease (HD) is an age-dependent neurodegenerative disease. DNA repair pathways have recently been implicated as the most predominant modifiers of age of onset in HD patients. We report that endogenous huntingtin protein directly participates in oxidative DNA damage repair. Using novel chromobodies to detect endogenous human huntingtin in live cells, we show that localization of huntingtin to DNA damage sites is dependent on the kinase activity of ataxia telangiectasia mutated (ATM) protein. Super-resolution microscopy and biochemical assays revealed that huntingtin co-localizes with and scaffolds proteins of the DNA damage response pathway in response to oxidative stress. In HD patient fibroblasts bearing typical clinical HD allele lengths, we demonstrate that there is deficient oxidative DNA damage repair. We propose that DNA damage in HD is caused by dysfunction of the mutant huntingtin protein in DNA repair, and accumulation of DNA oxidative lesions due to elevated reactive oxygen species may contribute to the onset of HD.

Huntingtin N17 domain is a reactive oxygen species sensor regulating huntingtin phosphorylation and localization.

The N17 domain of the huntingtin protein is post-translationally modified and is the master regulator of huntingtin intracellular localization. In Huntington's disease (HD), mutant huntingtin is hypo-phosphorylated at serines 13 and 16 within N17, and increasing N17 phosphorylation has been shown to be protective in HD mouse models. Thus, N17 phosphorylation is defined as a sub-target of huntingtin for potential therapeutic intervention. We have previously shown that cellular stress can affect huntingtin nuclear entry and phosphorylation. Here, we demonstrate that huntingtin localization can be specifically affected by reactive oxygen species (ROS) stress. We have located the sensor of this stress to the N17 domain, specifically to a highly conserved methionine at position 8. In vitro, we show by circular dichroism spectroscopy structural studies that the alpha-helical structure of N17 changes in response to redox conditions and show that the consequence of this change is enhanced N17 phosphorylation and nuclear targeting of endogenous huntingtin. Using N17 substitution point mutants, we demonstrate that N17 sulphoxidation enhances N17 dissociation from the endoplasmic reticulum (ER) membrane. This enhanced solubility makes N17 a better substrate for phosphorylation and subsequent nuclear retention. This ability of huntingtin to sense ROS levels at the ER, with phosphorylation and nuclear localization as a response, suggests that ROS stress due to aging could be a critical molecular trigger of huntingtin functions and dysfunctions in HD and may explain the age-onset nature of the disorder.

Polyglutamine domain flexibility mediates the proximity between flanking sequences in huntingtin.

Huntington disease (HD) is a neurodegenerative disorder caused by a CAG expansion within the huntingtin gene that encodes a polymorphic glutamine tract at the amino terminus of the huntingtin protein. HD is one of nine polyglutamine expansion diseases. The clinical threshold of polyglutamine expansion for HD is near 37 repeats, but the mechanism of this pathogenic length is poorly understood. Using Förster resonance energy transfer, we describe an intramolecular proximity between the N17 domain and the downstream polyproline region that flanks the polyglutamine tract of huntingtin. Our data support the hypothesis that the polyglutamine tract can act as a flexible domain, allowing the flanking domains to come into close spatial proximity. This flexibility is impaired with expanded polyglutamine tracts, and we can detect changes in huntingtin conformation at the pathogenic threshold for HD. Altering the structure of N17, either via phosphomimicry or with small molecules, also affects the proximity between the flanking domains. The structural capacity of N17 to fold back toward distal regions within huntingtin requires an interacting protein, protein kinase C and casein kinase 2 substrate in neurons 1 (PACSIN1). This protein has the ability to bind both N17 and the polyproline region, stabilizing the interaction between these two domains. We also developed an antibody-based FRET assay that can detect conformational changes within endogenous huntingtin in wild-type versus HD fibroblasts. Therefore, we hypothesize that wild-type length polyglutamine tracts within huntingtin can form a flexible domain that is essential for proper functional intramolecular proximity, conformations, and dynamics.

Identification of a karyopherin ß1/ß2 proline-tyrosine nuclear localization signal in huntingtin protein.

Among the known pathways of protein nuclear import, the karyopherin ß2/transportin pathway is only the second to have a defined nuclear localization signal (NLS) consensus. Huntingtin, a 350-kDa protein, has defined roles in the nucleus, as well as a CRM1/exportin-dependent nuclear export signal; however, the NLS and exact pathway of import have remained elusive. Here, using a live cell assay and affinity chromatography, we show that huntingtin has a karyopherin ß2-dependent proline-tyrosine (PY)-NLS in the amino terminus of the protein. This NLS comprises three consensus components: a basic charged sequence, a downstream conserved arginine, and a PY sequence. Unlike the classic PY-NLS, which has an unstructured intervening sequence between the consensus components, we show that a ß sheet structured region separating the consensus elements is critical for huntingtin NLS function. The huntingtin PY-NLS is also capable of import through the importin/karyopherin ß1 pathway but was not functional in all cell types tested. We propose that this huntingtin PY-NLS may comprise a new class of multiple import factor-dependent NLSs with an internal structural component that may regulate NLS activity.

Kinase inhibitors modulate huntingtin cell localization and toxicity.

Two serine residues within the first 17 amino acid residues of huntingtin (N17) are crucial for modulation of mutant huntingtin toxicity in cell and mouse genetic models of Huntington's disease. Here we show that the stress-dependent phosphorylation of huntingtin at Ser13 and Ser16 affects N17 conformation and targets full-length huntingtin to chromatin-dependent subregions of the nucleus, the mitotic spindle and cleavage furrow during cell division. Polyglutamine-expanded mutant huntingtin is hypophosphorylated in N17 in both homozygous and heterozygous cell contexts. By high-content screening in live cells, we identified kinase inhibitors that modulated N17 phosphorylation and hence huntingtin subcellular localization. N17 phosphorylation was reduced by casein kinase-2 inhibitors. Paradoxically, IKKß kinase inhibition increased N17 phosphorylation, affecting huntingtin nuclear and subnuclear localization. These data indicate that huntingtin phosphorylation at Ser13 and Ser16 can be modulated by small-molecule drugs, which may have therapeutic potential in Huntington's disease.

Huntingtin has a membrane association signal that can modulate huntingtin aggregation, nuclear entry and toxicity.

Huntington's disease is caused by an expanded polyglutamine tract in huntingtin protein, leading to accumulation of huntingtin in the nuclei of striatal neurons. The 18 amino-acid amino-terminus of huntingtin is an amphipathic alpha helical membrane-binding domain that can reversibly target to vesicles and the endoplasmic reticulum (ER). The association of huntingtin to the ER is affected by ER stress. A single point mutation in huntingtin 1-18 predicted to disrupt this helical structure displayed striking phenotypes of complete inhibition of polyglutamine-mediated aggregation, increased huntingtin nuclear accumulation and greatly increased mutant huntingtin toxicity in a striatal-derived mouse cell line. Huntingtin vesicular interaction mediated by 1-18 is specific to late endosomes and autophagic vesicles. We propose that huntingtin has a normal biological function as an ER-associated protein that can translocate to the nucleus and back out in response to ER stress or other events. The increased nuclear entry of mutant huntingtin due to loss of ER-targeting results in increased toxicity.

Canadian Association of Neurosciences Review: polyglutamine expansion neurodegenerative diseases.

Since the early 1990s, DNA triplet repeat expansions have been found to be the cause in an ever increasing number of genetic neurologic diseases. A subset of this large family of genetic diseases has the expansion of a CAG DNA triplet in the open reading frame of a coding exon. The result of this DNA expansion is the expression of expanded glutamine amino acid repeat tracts in the affected proteins, leading to the term, Polyglutamine Diseases, which is applied to this sub-family of diseases. To date, nine distinct genes are known to be linked to polyglutamine diseases, including Huntington's disease, Machado-Joseph Disease and spinobulbar muscular atrophy or Kennedy's disease. Most of the polyglutamine diseases are characterized clinically as spinocerebellar ataxias. Here we discuss recent successes and advancements in polyglutamine disease research, comparing these different diseases with a common genetic flaw at the level of molecular biology and early drug design for a family of diseases where many new research tools for these genetic disorders have been developed. Polyglutamine disease research has successfully used interdisciplinary collaborative efforts, informative multiple mouse genetic models and advanced tools of pharmaceutical industry research to potentially serve as the prototype model of therapeutic research and development for rare neurodegenerative diseases.

Practical three color live cell imaging by widefield microscopy.

Live cell fluorescence microscopy using fluorescent protein tags derived from jellyfish and coral species has been a successful tool to image proteins and dynamics in many species. Multi-colored aequorea fluorescent protein (AFP) derivatives allow investigators to observe multiple proteins simultaneously, but overlapping spectral properties sometimes require the use of sophisticated and expensive microscopes. Here, we show that the aequorea coerulescens fluorescent protein derivative, PS-CFP2 has excellent practical properties as a blue fluorophore that are distinct from green or red fluorescent proteins and can be imaged with standard filter sets on a widefield microscope. We also find that by widefield illumination in live cells, that PS-CFP2 is very photostable. When fused to proteins that form concentrated puncta in either the cytoplasm or nucleus, PSCFP2 fusions do not artifactually interact with other AFP fusion proteins, even at very high levels of over-expression. PSCFP2 is therefore a good blue fluorophore for distinct three color imaging along with eGFP and mRFP using a relatively simple and inexpensive microscope.

Ataxin-7 can export from the nucleus via a conserved exportin-dependent signal.

Spinocerebellar ataxia type 7 is a progressive neurodegenerative disorder caused by a CAG DNA triplet repeat expansion leading to an expanded polyglutamine tract in the ataxin-7 protein. Ataxin-7 appears to be a transcription factor and a component of the STAGA transcription coactivator complex. Here, using live cell imaging and inverted fluorescence recovery after photobleaching, we demonstrate that ataxin-7 has the ability to export from the nucleus via the CRM-1/exportin pathway and that ataxin-7 contains a classic leucine-type nuclear export signal (NES). We have precisely defined the location of this NES in ataxin-7 and found it to be fully conserved in all vertebrate species. Polyglutamine expansion was seen to reduce the nuclear export rate of mutant ataxin-7 relative to wild-type ataxin-7. Subtle point mutation of the NES in polyglutamine expanded ataxin-7 increased toxicity in primary cerebellar neurons in a polyglutamine length-dependent manner in the context of full-length ataxin-7. Our results add ataxin-7 to a growing list of polyglutamine disease proteins that are capable of nuclear shuttling, and we define an activity of ataxin-7 in the STAGA complex of trafficking between the nucleus and cytoplasm.

Huntingtin contains a highly conserved nuclear export signal.

Huntington's disease (HD), is a genetic neurodegenerative disease characterized by a DNA CAG triplet repeat expansion in the first exon of the disease gene, HD. CAG DNA expansion results in a polyglutamine tract expansion in mutant huntingtin protein. Wild-type and mutant full-length huntingtin have been detected in the nucleus, but elevated levels of mutant huntingtin and huntingtin amino-terminal proteolytic fragments are seen to accumulate in the nuclei of HD-affected neurons. The presence of huntingtin in both the nucleus and the cytoplasm suggested that huntingtin may be dynamic between these compartments. By live cell time-lapse video microscopy, we have been able to visualize polyglutamine-mediated aggregation and the transient nuclear localization of huntingtin over time in a striatal cell line. A classical nuclear localization signal could not be detected in huntingtin, but we have discovered a nuclear export signal (NES) in the carboxy-terminus of huntingtin. Leptomycin B treatment of clonal striatal cells enhanced the nuclear localization of huntingtin, and a mutant NES huntingtin displayed increased nuclear localization, indicating that huntingtin can shuttle to and from the nucleus. The huntingtin NES is strictly conserved among all huntingtin proteins from diverse species. This export signal may be important in Huntington's disease because this fragment of huntingtin is proteolytically cleaved away during HD. The huntingtin NES therefore defines a potential role for huntingtin as a member of a nucleocytoplasmic dynamic protein complex.