Glutamine Stretch Research Articles

Characterization of immunoglobulin-ε (IgE) to benefit therapeutic IgE engineering.

Several food proteins, e.g. in peanut, can trigger adversely immune responses that lead to severe, even fatal, clinical symptoms. Immunoglobulin-ε (IgE) is the main immunoglobulin isotype associated with such Type-I hypersensitivity. The allergic immune reactions are mediated by IgE and its interactions to the receptor FcεRIα located on mast cells. The cellular activation is triggered by the FcεRIα-bound IgE typically with multivalent antigen binding or occasionally without any presence of known antigens. Disrupting such interactions could intervene the allergic immune responses.Our previous study (doi: 10.1016/j.jaci.2019.03.028) suggested prolonged FcεRIα-IgE interactions led to emergence and over-presentation of the persistent IgE. Such effects were due to long-ranged communications between the IgE V-regions and the FcεRIα-binding site. Additionally, varying structural arrangements of the IgE complementarity-determining regions (CDRs) and framework regions (FWRs) of both the antibody heavy and light chains with influence from the CH-region could orient a single mutation in the VH-CDR2 to abrogate the non-specific binding of superantigen spA or could form inducible structural glutamine stretches that bound nickel Ni2+ without losing the antigen specificity (doi: 10.3389/fimmu.2021.676048). In fact, these work advocate analyzing IgE holistically as a whole, rather than a sum of its parts.In the current work riding on the long-ranged communications between the IgE antigen-binding and FcεRIα-binding regions, we further characterize the impact of the linking Cε2 on the IgE conformational changes upon antigen and/or FcεRIα binding, applied in peanut allergy. These characteristics elucidate the structural mechanisms for allosteric effects observed in the allergen and the FcεRIα binding. This will provide better understanding of initiating steps in the Type-I hypersensitivity as well as the crucial interdomain communications in the whole IgE structure, and hence further complement understanding the role of IgE in allergy pathogenesis.

Molecular Insights of Nickel Binding to Therapeutic Antibodies as a Possible New Antibody Superantigen

The binding of nickel by immune proteins can manifest as Type IV contact dermatitis (Ni-specific T cells mediated) and less frequently as Type I hypersensitivity with both mechanisms remaining unknown to date. Since there are reports of patients co-manifesting the two hypersensitivities, a common mechanism may underlie both the TCR and IgE nickel binding. Focusing on Trastuzumab and Pertuzumab IgE variants as serendipitous investigation models, we found Ni-NTA interactions independent of Her2 binding to be due to glutamine stretches. These stretches are both Ni-inducible and in fixed pockets at the antibody complementarity-determining regions (CDRs) and framework regions (FWRs) of both the antibody heavy and light chains with influence from the heavy chain constant region. Comparisons with TCRs structures revealed similar interactions, demonstrating the possible underlying mechanism in selecting for Ni-binding IgEs and TCRs respectively. With the elucidation of the interaction, future therapeutic antibodies could also be sagaciously engineered to utilize such nickel binding for biotechnological purposes.

The phasor FLIM method reveals a link between a change in energy metabolism and mHtt protein spread in healthy Mammalian cells when co-cultured with Huntington diseased cells

Huntington Disease (HD) is a late-onset autosomal neurodegenerative disease characterized by the aggregations of mutant Huntingtin proteins (mHTT). A glutamine stretch (PolyQ) at the N-terminal of the Huntingtin protein is generated by the abnormal expansion of CAG trinucleotide repeats in exon 1 of the HTT gene. While the resulting polyQ aggregates are the predominate feature of HD, the intercellular spread of the expanded protein and the effect upon this transfer inside healthy cells have not yet fully understood. Here, we have employed the phasor Fluorescence Lifetime Imaging Microscopy (FLIM) method to measure NADH fluorescence lifetime change after the internalization of the PolyQ protein. Based on our analysis, we have found a significant decrease in the fraction of bound NADH in both cytoplasmic and nucleus regions when cells are co-cultured or when healthy cells uptake the supernatant containing polyQ proteins and aggregates. Overall, our FLIM study combined with confocal fluorescence imaging visualizes the absorption of the mutant Htt protein aggregates which results in a distinct NADH fluorescence lifetime between control cells and acceptor cells. These studies show, for the first time, the influence of how neighboring cells expressing the expanded Htt protein can regulate energy metabolism in healthy cells.

Huntingtin Maintains Mitochondrial Genome Integrity and Function

SUMMARYThe function of huntingtin (HTT) is enigmatic in that the native protein provides neuroprotection while mutant HTT (mHTT) carrying an expanded stretch of glutamines triggers neurotoxicity in Huntington’s disease (HD). We recently reported that HTT forms a transcription-coupled DNA repair (TCR) complex with RNA polymerase and DNA repair enzyme polynucleotide-kinase-3'-phosphatase (PNKP). This complex resolves DNA lesions during transcription to maintain genome integrity, while mHTT impairs the activity of this complex, resulting in DNA lesion accumulation, activating the DNA damage-response in HD. Here we report that HTT forms a DNA repair complex within the mitochondria with mitochondrial RNA polymerase, transcription factors, and PNKP. This complex resolves mitochondrial DNA (mtDNA) lesions to preserve mtDNA integrity and function. Pathogenic mHTT reduces the complex activity, resulting in persistence of mtDNA lesions, impairing mitochondrial function in HD. Restoring the activity of the mtDNA repair complex in a Drosophila model of HD dramatically improves mtDNA integrity, and motor defects.

Blattella germanica displays a large arsenal of antimicrobial peptide genes

Defence systems against microbial pathogens are present in most living beings. The German cockroach Blattella germanica requires these systems to adapt to unhealthy environments with abundance of pathogenic microbes, in addition to potentially control its symbiotic systems. To handle this situation, four antimicrobial gene families (defensins, termicins, drosomycins and attacins) were expanded in its genome. Remarkably, a new gene family (blattellicins) emerged recently after duplication and fast evolution of an attacin gene, which is now encoding larger proteins with the presence of a long stretch of glutamines and glutamic acids. Phylogenetic reconstruction, within Blattellinae, suggests that this duplication took place before the divergence of Blattella and Episymploce genera. The latter harbours a long attacin gene (pre-blattellicin), but the absence of the encoded Glx-region suggests that this element evolved recently in the Blattella lineage. A screening of AMP gene expression in available transcriptomic SR projects of B. germanica showed that, while some AMPs are expressed during almost the whole development, others are restricted to shorter periods. Blattellicins are highly expressed only in adult females. None of the available SR tissue projects could be associated with blattellicins’ expression, suggesting that it takes place in other tissues, maybe the gut.

Ataxin-1 regulates B cell function and the severity of autoimmune experimental encephalomyelitis

Ataxin-1 (ATXN1) is a ubiquitous polyglutamine protein expressed primarily in the nucleus where it binds chromatin and functions as a transcriptional repressor. Mutant forms of ataxin-1 containing expanded glutamine stretches cause the movement disorder spinocerebellar ataxia type 1 (SCA1) through a toxic gain-of-function mechanism in the cerebellum. Conversely, ATXN1 loss-of-function is implicated in cancer development and Alzheimer's disease (AD) pathogenesis. ATXN1 was recently nominated as a susceptibility locus for multiple sclerosis (MS). Here, we show that Atxn1-null mice develop a more severe experimental autoimmune encephalomyelitis (EAE) course compared to wildtype mice. The aggravated phenotype is mediated by increased T helper type 1 (Th1) cell polarization, which in turn results from the dysregulation of B cell activity. Ataxin-1 ablation in B cells leads to aberrant expression of key costimulatory molecules involved in proinflammatory T cell differentiation, including cluster of differentiation (CD)44 and CD80. In addition, comprehensive phosphoflow cytometry and transcriptional profiling link the exaggerated proliferation of ataxin-1 deficient B cells to the activation of extracellular signal-regulated kinase (ERK) and signal transducer and activator of transcription (STAT) pathways. Lastly, selective deletion of the physiological binding partner capicua (CIC) demonstrates the importance of ATXN1 native interactions for correct B cell functioning. Altogether, we report a immunomodulatory role for ataxin-1 and provide a functional description of the ATXN1 locus genetic association with MS risk.

In Vivo Quantification of Protein Turnover in Aging C. Elegans using Photoconvertible Dendra2.

Proteins are synthesized and degraded constantly within a cell to maintain homeostasis. Being able to monitor the degradation of a protein of interest is key to understanding not only its life cycle, but also to uncover imbalances in the proteostasis network. This method shows how to track the degradation of the disease-causing protein huntingtin. Two versions of huntingtin fused to Dendra2 are expressed in the C. elegans nervous system: a physiological version or one with an expanded and pathogenic stretch of glutamines. Dendra2 is a photoconvertible fluorescent protein; upon a short ultraviolet (UV) irradiation pulse, Dendra2 switches its excitation/emission spectra from green to red. Similar to a pulse-chase experiment, the turnover of the converted red-Dendra2 can be monitored and quantified, regardless of the interference from newly synthesized green-Dendra2. Using confocal-based microscopy and due to the optical transparency of C. elegans, it is possible to monitor and quantify the degradation of huntingtin-Dendra2 in a living, aging organism. Neuronal huntingtin-Dendra2 is partially degraded soon after conversion and cleared further over time. The systems controlling degradation are deficient in the presence of mutant huntingtin and are further impaired with aging. Neuronal subtypes within the same nervous system exhibit different turnover capacities for huntingtin-Dendra2. Overall, monitoring any protein of interest fused to Dendra2 can provide important information not only on its degradation and the players of the proteostasis network involved, but also on its location, trafficking, and transport.

Effects of flanking sequences and cellular context on subcellular behavior and pathology of mutant HTT.

Huntington's disease (HD) is caused by an expansion of a poly glutamine (polyQ) stretch in the huntingtin protein (HTT) that is necessary to cause pathology and formation of HTT aggregates. Here we ask whether expanded polyQ is sufficient to cause pathology and aggregate formation. By addressing the sufficiency question, one can identify cellular processes and structural parameters that influence HD pathology and HTT subcellular behavior (i.e. aggregation state and subcellular location). Using Drosophila, we compare the effects of expressing mutant full-length human HTT (fl-mHTT) to the effects of mutant human HTTexon1 and to two commonly used synthetic fragments, HTT171 and shortstop (HTT118). Expanded polyQ alone is not sufficient to cause inclusion formation since full-length HTT and HTTex1 with expanded polyQ are both toxic although full-length HTT remains diffuse while HTTex1 forms inclusions. Further, inclusions are not sufficient to cause pathology since HTT171-120Q forms inclusions but is benign and co-expression of HTT171-120Q with non-aggregating pathogenic fl-mHTT recruits fl-mHTT to aggregates and rescues its pathogenicity. Additionally, the influence of sequences outside the expanded polyQ domain is revealed by finding that small modifications to the HTT118 or HTT171 fragments can dramatically alter their subcellular behavior and pathogenicity. Finally, mutant HTT subcellular behavior is strongly modified by different cell and tissue environments (e.g. fl-mHTT appears as diffuse nuclear in one tissue and diffuse cytoplasmic in another but toxic in both). These observations underscore the importance of cellular and structural context for the interpretation and comparison of experiments using different fragments and tissues to report the effects of expanded polyQ.

Neuroprotective Effects of N - Acetyl - Methoxytryptamine in a Neurotoxic Mice Model of Huntington’s Disease Induced by Quinolinic Acid

Huntington’s Disease (HD) is an autosomal dominant neuropathology associated with severe degeneration in neurons of the basal ganglia. It is characterized by progressive motors symptoms, cognitive deficits and dementia. The genetic mutation induces trinucl eotide repeat expansion (TNR) of cytosine, adenine and guanine (CAG). The CAG - repeat is translated into a poly glutamine (polyQ) stretch caused by molding of DNA “loop out” structures and DNA slippage during the replication. This expanded sequence encodes a mutant huntingtin (mHtt), which is associated to neuronal dysfunction, cellular death, ubiquitous molecular and cellular anomalies. Furthermore, there are progressive impairments in motor control, cognitive function and mood. Quinolinic acid (QA) is an e xcitotoxic compound in humans and animals that has been linked to HD. N - acetyl - methoxytryptamine (MLT) is a hormone that produce anti - oxidant and anti - inflammatory effects on cell damage. The principal aim of this study is to know if QA induces damage in s triatal astrocytes and neurons and to evidence how MLT can participate as a neuroprotectant agent against the neurotoxic injury. We inoculate QA intrastriatal in 8 weeks old wild type (WT) female mice as endogenous neurotoxic model of HD. Here, we worked w ith three different groups of mice, and the striatal injection was administrated in all animals. PBS were inoculated in control mice; the second group was injected only QA; and the last group was pre - treated with MLT 30 min before the QA injection. Immunob lots, oxidative stress biomarkers, histological assay of astrocytes and neurons were evaluated as well as rotarod and open field tests. There was a reduction in proteins levels of BDNF and NeuN in QA mice. Contrary effects in lysates showed NF - κB, IκB α , HT T, GFAP and NRs. The analysis of CAT, SOD, Gpx and GSH showed decreased activity, while MDA displayed increased activation in QA mice. The animals pre - treated with MLT revers the tendency of proteins and antioxidant biomarkers; the HTT perinuclear staining in neurons was reduced. NeuN histochemistry showed a greater number of NeuN + cells; the astrogliosis was attenuated; and locomotor activities improved. Analysis of RT - PCR do no show significant diferences between teatments. This study suggests that MLT c an induce a compensatory response to neurotoxic stress againts neuro - glial injury. Thus, increasing MLT levels may be a promising treatment for HD disorder.

Glutamine Codon Usage and polyQ Evolution in Primates Depend on the Q Stretch Length.

Amino acid usage in a proteome depends mostly on its taxonomy, as it does the codon usage in transcriptomes. Here, we explore the level of variation in the codon usage of a specific amino acid, glutamine, in relation to the number of consecutive glutamine residues. We show that CAG triplets are consistently more abundant in short glutamine homorepeats (polyQ, four to eight residues) than in shorter glutamine stretches (one to three residues), leading to the evolutionary growth of the repeat region in a CAG-dependent manner. The length of orthologous polyQ regions is mostly stable in primates, particularly the short ones. Interestingly, given a short polyQ the CAG usage is higher in unstable-in-length orthologous polyQ regions. This indicates that CAG triplets produce the necessary instability for a glutamine stretch to grow. Proteins related to polyQ-associated diseases behave in a more extreme way, with longer glutamine stretches in human and evolutionarily closer nonhuman primates, and an overall higher CAG usage. In the light of our results, we suggest an evolutionary model to explain the glutamine codon usage in polyQ regions.

Huntingtin Is Required for Neural But Not Cardiac/Pancreatic Progenitor Differentiation of Mouse Embryonic Stem Cells In vitro.

Mutation in the huntingtin (HTT) gene causes Huntington’s disease (HD). It is an autosomal dominant trinucleotide-repeat expansion disease in which CAG repeat sequence expands to >35. This results in the production of mutant HTT protein with an increased stretch of glutamines near the N-terminus. The wild type HTT gene encodes a 350 kD protein whose function remains elusive. Mutant HTT protein has been implicated in transcription, axonal transport, cytoskeletal structure/function, signal transduction, and autophagy. HD is characterized by the appearance of nuclear inclusions and degeneration of the striatum. Although HTT protein is expressed early in embryos, most patients develop symptoms in mid-life. It is also unclear why the ubiquitously expressed mutant HTT specifically causes striatal atrophy. Wild type Htt is essential for development as Htt knockout mice die at day E7.5. Increasing evidence suggests mutant Htt may alter neurogenesis and development of striatal neurons resulting in neuronal loss. Using a mouse embryonic stem cell model, we examined the role of Htt in neural differentiation. We found cells lacking Htt inefficient in generating neural stem cells. In contrast differentiation into progenitors of mesoderm and endoderm lineages was not affected. The data suggests Htt is essential for neural but not cardiac/pancreatic progenitor differentiation of embryonic stem cells in vitro.

Modulation of mutant Huntingtin aggregates and toxicity by human myeloid leukemia factors

Increased poly glutamine (polyQ) stretch at N-terminal of Huntingtin (HTT) causes Huntington’s disease. HTT interacts with large number of proteins, although the preference for such interactions with wild type or mutated HTT protein remains largely unknown. HYPK, an intrinsically unstructured protein chaperone and interactor of mutant HTT was found to interact with myeloid leukemia factor 1 (MLF1) and 2 (MLF2). To identify the role of these two proteins in mutant HTT mediated aggregate formation and toxicity in a cell model, both the proteins were found to preferentially interact with the mutated N-terminal HTT. They significantly reduced the number of cells containing mutant HTT aggregates and subsequent apoptosis in Neuro2A cells. Additionally, in FRAP assay, mobile fraction of mutant HTT aggregates was increased in the presence of MLF1 or MLF2. Further, MLF1 could release transcription factors like p53, CBP and CREB from mutant HTT aggregates. Moreover, in HeLa cell co-expressing mutant HTT exon1 and full length MLF1, p53 was released from the aggregates, leading to the recovery of the expression of the GADD45A transcript, a p53 regulated gene. Taking together, these results showed that MLF1 and MLF2 modulated the formation of aggregates and induction of apoptosis as well as the expressions of genes indirectly.

A triazole derivative elicits autophagic clearance of polyglutamine aggregation in neuronal cells

Trinucleotide CAG repeat expansion in the coding region of genes has a propensity to form polyglutamine (polyQ) aggregates that contribute to neuronal disorders. Strategies in elevating autophagy to disintegrate the insoluble aggregates without injuring cells have become a major goal for therapy. In this work, a triazole derivative, OC-13, was found accelerating autophagic clearance of polyQ aggregation in human neuroblastoma cells following induction of the enhanced green fluorescence-conjugated chimeric protein that enclosed 79 polyQ repeats (Q79-EGFP). OC-13 accelerated autophagy development and removed nuclear Q79-EGFP aggregates. The increase of Beclin-1, turnover of LC3-I to LC3-II and degradation of p62 supported autophagy activation. Pretreatment of autophagy inhibitor, bafilomycin A1, not only suppressed autophagolysome fusion, but also impeded aggregate eradication. The study also showed that c-Jun N-terminal kinase/Beclin-1 pathway was activated during OC-13 treatment and c-Jun N-terminal kinase inhibitor impaired autophagy and final breakdown. Autophagic clearance of the insoluble aggregates demonstrated the feasibility of OC-13 in alleviating neuronal disorders because of expanded glutamine stretches.

Association of status redox with demographic, clinical and imaging parameters in patients with Huntington's disease

Huntington's disease (HD) is an autosomal dominant, progressive neurodegenerative disorder, caused by an expanded trinucleotide CAG sequence of the huntingtin (Htt) gene, which encodes a stretch of glutamines in the Htt protein. The mechanisms of neurodegeneration associated with the accumulation of Htt aggregates still remains unclear. ObjectivesTo determine oxidative stress biomarkers in HD patients and their relationship with clinical, demographic and neuroimaging parameters. Design and methodsFourteen patients and 39 controls paired by age and sex participated in this study. Oxidative damage was assayed in blood by measuring malondialdehyde (MDA) and advanced oxidative protein products (AOPPs). Antioxidant status was determined by activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), reduced glutathione (GSH), protein thiols and total antioxidant capacity (FRAP). The Unified Huntington Disease Rating Scale (UHDRS) and neuroimaging studies were also employed. ResultsMDA, AOPP and GPx were significantly increased in HD patients with respect to the control group, while GR activity was decreased. FRAP correlated with age of disease onset, AOPP with motor severity (UHDRS score), age of patients and age of disease onset. Caudate atrophy was associated with lower plasma concentrations of GSH. ConclusionsThese findings point to a redox imbalance in HD patients. GR activity could be a potential biomarker for symptom onset in asymptomatic gene carriers, while plasmatic GSH could be useful in monitoring the progression of neurodegeneration – as an expression of caudate atrophy – during the course of the disease.

Viral vector mediated expression of mutant huntingtin in the dorsal raphe produces disease-related neuropathology but not depressive-like behaviors in wildtype mice

Huntington׳s disease (HD) is a neurodegenerative disorder caused by a mutation in the HTT gene (mHTT) encoding the protein huntingtin. An expansion in the gene׳s CAG repeat length renders a misfolded, dysfunctional protein with an abnormally long glutamine (Q) stretch at the N terminus that often incorporates into inclusion bodies and leads to neurodegeneration in many regions of the brain. HD is characterized by motor and cognitive decline as well as mood disorders, with depression being particularly common. Approximately 40% of the HD population suffers from depressive symptoms. Because these symptoms often manifest a decade or more prior to the knowledge that the person is at risk for the disease, a portion of the early depression in HD appears to be a consequence of the pathology arising from expression of the mutant gene. While the depression in HD patients is often treated with serotonin agonists, there is scant experimental evidence that the depression in HD responds well to these serotonin treatments or in a similar manner to how non-HD depression tends to respond. Additionally, at very early sub-threshold depression levels, abnormal changes in several neuronal populations are already detectable in HD patients, suggesting that a variety of brain structures may be involved. Taken together, the serotonin system is a viable candidate. However, at present there is limited evidence of the precise nuclei or circuits that play a role in HD depression. With this in mind, the current study was designed to control for the widespread brain neuropathology that occurs in HD and in transgenic mouse models of HD and focuses specifically on the influence of the midbrain dorsal raphe nucleus (DRN). The DRN provides the majority of the serotonin to the forebrain and exhibits cell loss in non-HD depression. Therefore, we employed a viral vector delivery system to investigate whether the over-expression of mHTT in the DRN׳s ventral sub-nuclei alone is sufficient to produce depressive-like behaviors. Wildtype mice were injected with an adeno-associated virus (AAV2/1) encoding HTT containing either a pathogenic (N171-82Q) or control (N171-16Q) CAG repeat length into the ventral DRN and depressive-like behaviors and motor behaviors were assessed for 12 weeks post-surgery. Quantitative PCR and immunohistochemistry (IHC) verified positive transduction in the ventral aspects of the DRN, including the ventral sub-nucleus (DRv) and interfascicular sub-nucleus (DRif). IHC demonstrated microgliosis in and around the injection site and mHTT-positive inclusions in serotonin-producing neurons and a small percentage of astrocytes in animals injected with N171-82Q compared to controls. Moreover, N171-82Q injected mice showed a 75% reduction in cells that stained positive for the serotonin synthesis enzyme, tryptophan hydroxylase-2 (TPH2) compared to controls (p<0.05). Despite mHTT-mediated pathology in the DRv and DRif, no significant changes in depressive-like behavior were detected. Consequently, we conclude that 12 weeks of N171-82Q expression in the ventral sub-nuclei of the DRN of wildtype mice causes characteristic disease-related cellular neuropathology but is not sufficient to elicit depressive-like behaviors. Ongoing studies are investigating whether a larger injection volume that transfects a larger percentage of the DRN and/or a longer time course of mHTT expression might elicit depressive-like behaviors. Moreover, mHTT expression in other regions of the brain, such as the hippocampal dentate gyrus and/or the frontal cortex might be necessary to elicit HD depression. Together, these results may prove helpful in addressing which therapeutic and/or pharmacological strategies might be most efficacious when treating depressive symptomology in patients suffering from HD.

High-resolution genetic mapping of allelic variants associated with cell wall chemistry in Populus.

BackgroundQTL cloning for the discovery of genes underlying polygenic traits has historically been cumbersome in long-lived perennial plants like Populus. Linkage disequilibrium-based association mapping has been proposed as a cloning tool, and recent advances in high-throughput genotyping and whole-genome resequencing enable marker saturation to levels sufficient for association mapping with no a priori candidate gene selection. Here, multiyear and multienvironment evaluation of cell wall phenotypes was conducted in an interspecific P. trichocarpa x P. deltoides pseudo-backcross mapping pedigree and two partially overlapping populations of unrelated P. trichocarpa genotypes using pyrolysis molecular beam mass spectrometry, saccharification, and/ or traditional wet chemistry. QTL mapping was conducted using a high-density genetic map with 3,568 SNP markers. As a fine-mapping approach, chromosome-wide association mapping targeting a QTL hot-spot on linkage group XIV was performed in the two P. trichocarpa populations. Both populations were genotyped using the 34 K Populus Infinium SNP array and whole-genome resequencing of one of the populations facilitated marker-saturation of candidate intervals for gene identification.ResultsFive QTLs ranging in size from 0.6 to 1.8 Mb were mapped on linkage group XIV for lignin content, syringyl to guaiacyl (S/G) ratio, 5- and 6-carbon sugars using the mapping pedigree. Six candidate loci exhibiting significant associations with phenotypes were identified within QTL intervals. These associations were reproducible across multiple environments, two independent genotyping platforms, and different plant growth stages. cDNA sequencing for allelic variants of three of the six loci identified polymorphisms leading to variable length poly glutamine (PolyQ) stretch in a transcription factor annotated as an ANGUSTIFOLIA C-terminus Binding Protein (CtBP) and premature stop codons in a KANADI transcription factor as well as a protein kinase. Results from protoplast transient expression assays suggested that each of the polymorphisms conferred allelic differences in the activation of cellulose, hemicelluloses, and lignin pathway marker genes.ConclusionThis study illustrates the utility of complementary QTL and association mapping as tools for gene discovery with no a priori candidate gene selection. This proof of concept in a perennial organism opens up opportunities for discovery of novel genetic determinants of economically important but complex traits in plants.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1215-z) contains supplementary material, which is available to authorized users.

A Unique Conformational Behaviour of Glutamine Peptides

Trinucleotide repeat expansions (CAG) lead to increase in glutamine residues and hence increase in glutamine stretch. This leads to number of neurodegenerative diseases. Therefore, the conformation of poly Q of varying chain lengths has been investigated by quantum mechanical and molecular dynamics approaches. Glutamine contains amide linkage in the side chain. It is the interaction between side chain amide linkage and the peptide bond of the backbone which dictates the conformational behaviour. Some of the glutamine residues adopt phi psi values corresponding to poly-L-proline type II structure. Not more than three glutamine residues are found to have the same set of values and hence polyglutamine is adopting random coil structure. Carbonyl-carbonyl, CH-O interactions and hydrogen bond formation involving backbone and side chain amide chain linkages are found to contribute to the stability of the adopted structure. In simulation studies due to interaction of water molecules with the amide linkages the values undergo change and this leads to weakening of carbonyl-carbonyl interactions and hydrogen bonds. The conformational behaviour of polyglutamine peptides is shown to be chain length dependent and this may provide some insight regarding the aggregation behaviour of proteins containing poly Q stretch. Possibly this is the first systematic study of the conformational behaviour of polyglutamine peptides.

The Hsp70/90 cochaperone, Sti1, suppresses proteotoxicity by regulating spatial quality control of amyloid-like proteins

Conformational diseases are associated with the conversion of normal proteins into aggregation-prone toxic conformers with structures similar to that of β-amyloid. Spatial distribution of amyloid-like proteins into intracellular quality control centers can be beneficial, but cellular mechanisms for protective aggregation remain unclear. We used a high-copy suppressor screen in yeast to identify roles for the Hsp70 system in spatial organization of toxic polyglutamine-expanded Huntingtin (Huntingtin with 103Q glutamine stretch [Htt103Q]) into benign assemblies. Under toxic conditions, Htt103Q accumulates in unassembled states and speckled cytosolic foci. Subtle modulation of Sti1 activity reciprocally affects Htt toxicity and the packaging of Htt103Q into foci. Loss of Sti1 exacerbates Htt toxicity and hinders foci formation, whereas elevation of Sti1 suppresses Htt toxicity while organizing small Htt103Q foci into larger assemblies. Sti1 also suppresses cytotoxicity of the glutamine-rich yeast prion [RNQ+] while reorganizing speckled Rnq1-monomeric red fluorescent protein into distinct foci. Sti1-inducible foci are perinuclear and contain proteins that are bound by the amyloid indicator dye thioflavin-T. Sti1 is an Hsp70 cochaperone that regulates the spatial organization of amyloid-like proteins in the cytosol and thereby buffers proteotoxicity caused by amyloid-like proteins.

Ataxin-3 protein modification as a treatment strategy for spinocerebellar ataxia type 3: Removal of the CAG containing exon

Spinocerebellar ataxia type 3 is caused by a polyglutamine expansion in the ataxin-3 protein, resulting in gain of toxic function of the mutant protein. The expanded glutamine stretch in the protein is the result of a CAG triplet repeat expansion in the penultimate exon of the ATXN3 gene. Several gene silencing approaches to reduce mutant ataxin-3 toxicity in this disease aim to lower ataxin-3 protein levels, but since this protein is involved in deubiquitination and proteasomal protein degradation, its long-term silencing might not be desirable. Here, we propose a novel protein modification approach to reduce mutant ataxin-3 toxicity by removing the toxic polyglutamine repeat from the ataxin-3 protein through antisense oligonucleotide-mediated exon skipping while maintaining important wild type functions of the protein. In vitro studies showed that exon skipping did not negatively impact the ubiquitin binding capacity of ataxin-3. Our in vivo studies showed no toxic properties of the novel truncated ataxin-3 protein. These results suggest that exon skipping may be a novel therapeutic approach to reduce polyglutamine-induced toxicity in spinocerebellar ataxia type 3.

A network of genes connects polyglutamine toxicity to ploidy control in yeast

Neurodegeneration is linked to protein aggregation in several human disorders. In Huntington’s disease, the length of a polyglutamine stretch in Huntingtin is correlated to neuronal death. Here we utilize a model based on glutamine stretches of 0, 30 or 56 residues in Saccharomyces cerevisiae to understand how such toxic proteins interfere with cellular physiology. A toxicity-mimicking cytostatic effect is evident from compromised colony formation upon expression of polyglutamines. Interestingly, diploid cells are insensitive to polyglutamines and haploid cells can escape cytostasis by hyperploidization. Using a genome-wide screen for genes required to obtain the cytostatic effect, we identify a network related to the budding process and cellular division. We observe a striking mislocalization of the septins Cdc10 and Shs1 in cells arrested by polyglutamines, suggesting that the septin ring may be a pivotal structure connecting polyglutamine toxicity and ploidy.