Biochemistry and Pharmacology - Research Publications
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ItemTadpole-like Conformations of Huntingtin Exon 1 Are Characterized by Conformational Heterogeneity that Persists regardless of Polyglutamine Length(ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD, 2018-05-11)Soluble huntingtin exon 1 (Httex1) with expanded polyglutamine (polyQ) engenders neurotoxicity in Huntington's disease. To uncover the physical basis of this toxicity, we performed structural studies of soluble Httex1 for wild-type and mutant polyQ lengths. Nuclear magnetic resonance experiments show evidence for conformational rigidity across the polyQ region. In contrast, hydrogen-deuterium exchange shows absence of backbone amide protection, suggesting negligible persistence of hydrogen bonds. The seemingly conflicting results are explained by all-atom simulations, which show that Httex1 adopts tadpole-like structures with a globular head encompassing the N-terminal amphipathic and polyQ regions and the tail encompassing the C-terminal proline-rich region. The surface area of the globular domain increases monotonically with polyQ length. This stimulates sharp increases in gain-of-function interactions in cells for expanded polyQ, and one of these interactions is with the stress-granule protein Fus. Our results highlight plausible connections between Httex1 structure and routes to neurotoxicity.
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ItemA thiol probe for measuring unfolded protein load and proteostasis in cells(NATURE PUBLISHING GROUP, 2017-09-07)When proteostasis becomes unbalanced, unfolded proteins can accumulate and aggregate. Here we report that the dye, tetraphenylethene maleimide (TPE-MI) can be used to measure cellular unfolded protein load. TPE-MI fluorescence is activated upon labelling free cysteine thiols, normally buried in the core of globular proteins that are exposed upon unfolding. Crucially TPE-MI does not become fluorescent when conjugated to soluble glutathione. We find that TPE-MI fluorescence is enhanced upon reaction with cellular proteomes under conditions promoting accumulation of unfolded proteins. TPE-MI reactivity can be used to track which proteins expose more cysteine residues under stress through proteomic analysis. We show that TPE-MI can report imbalances in proteostasis in induced pluripotent stem cell models of Huntington disease, as well as cells transfected with mutant Huntington exon 1 before the formation of visible aggregates. TPE-MI also detects protein damage following dihydroartemisinin treatment of the malaria parasites Plasmodium falciparum. TPE-MI therefore holds promise as a tool to probe proteostasis mechanisms in disease.Proteostasis is maintained through a number of molecular mechanisms, some of which function to protect the folded state of proteins. Here the authors demonstrate the use of TPE-MI in a fluorigenic dye assay for the quantitation of unfolded proteins that can be used to assess proteostasis on a cellular or proteome scale.
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ItemA biosensor-based framework to measure latent proteostasis capacity(NATURE PUBLISHING GROUP, 2018-01-18)The pool of quality control proteins (QC) that maintains protein-folding homeostasis (proteostasis) is dynamic but can become depleted in human disease. A challenge has been in quantitatively defining the depth of the QC pool. With a new biosensor, flow cytometry-based methods and mathematical modeling we measure the QC capacity to act as holdases and suppress biosensor aggregation. The biosensor system comprises a series of barnase kernels with differing folding stability that engage primarily with HSP70 and HSP90 family proteins. Conditions of proteostasis stimulation and stress alter QC holdase activity and aggregation rates. The method reveals the HSP70 chaperone cycle to be rate limited by HSP70 holdase activity under normal conditions, but this is overcome by increasing levels of the BAG1 nucleotide exchange factor to HSPA1A or activation of the heat shock gene cluster by HSF1 overexpression. This scheme opens new paths for biosensors of disease and proteostasis systems.
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ItemNo Preview AvailableHuntingtin Inclusions Trigger Cellular Quiescence, Deactivate Apoptosis, and Lead to Delayed Necrosis(CELL PRESS, 2017-05-02)Competing models exist in the literature for the relationship between mutant Huntingtin exon 1 (Httex1) inclusion formation and toxicity. In one, inclusions are adaptive by sequestering the proteotoxicity of soluble Httex1. In the other, inclusions compromise cellular activity as a result of proteome co-aggregation. Using a biosensor of Httex1 conformation in mammalian cell models, we discovered a mechanism that reconciles these competing models. Newly formed inclusions were composed of disordered Httex1 and ribonucleoproteins. As inclusions matured, Httex1 reconfigured into amyloid, and other glutamine-rich and prion domain-containing proteins were recruited. Soluble Httex1 caused a hyperpolarized mitochondrial membrane potential, increased reactive oxygen species, and promoted apoptosis. Inclusion formation triggered a collapsed mitochondrial potential, cellular quiescence, and deactivated apoptosis. We propose a revised model where sequestration of soluble Httex1 inclusions can remove the trigger for apoptosis but also co-aggregate other proteins, which curtails cellular metabolism and leads to a slow death by necrosis.
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ItemN-Terminal Fragments of Huntingtin Longer than Residue 170 form Visible Aggregates Independently to Polyglutamine Expansion(IOS PRESS, 2017-01-01)BACKGROUND: A hallmark of Huntington's disease is the progressive aggregation of full length and N-terminal fragments of polyglutamine (polyQ)-expanded Huntingtin (Htt) into intracellular inclusions. The production of N-terminal fragments appears important for enabling pathology and aggregation; and hence the direct expression of a variety of N-terminal fragments are commonly used to model HD in animal and cellular models. OBJECTIVE: It remains unclear how the length of the N-terminal fragments relates to polyQ - mediated aggregation. We investigated the fundamental intracellular aggregation process of eight different-length N-terminal fragments of Htt in both short (25Q) and long polyQ (97Q). METHODS: N-terminal fragments were fused to fluorescent proteins and transiently expressed in mammalian cell culture models. These included the classic exon 1 fragment (90 amino acids) and longer forms of 105, 117, 171, 513, 536, 552, and 586 amino acids based on wild-type Htt (of 23Q) sequence length nomenclature. RESULTS: N-terminal fragments of less than 171 amino acids only formed inclusions in polyQ-expanded form. By contrast the longer fragments formed inclusions irrespective of Q-length, with Q-length playing a negligible role in extent of aggregation. The inclusions could be classified into 3 distinct morphological categories. One type (Type A) was universally associated with polyQ expansions whereas the other two types (Types B and C) formed independently of polyQ length expansion. CONCLUSIONS: PolyQ-expansion was only required for fragments of less than 171 amino acids to aggregate. Longer fragments aggregated predominately through a non-polyQ mechanism, involving at least one, and probably more distinct clustering mechanisms.
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ItemWhen Proteostasis Goes Bad: Protein Aggregation in the Cell(WILEY, 2017-02-01)Protein aggregation is a hallmark of the major neurodegenerative diseases including Alzheimer's, Parkinson's, Huntington's and motor neuron and is a symptom of a breakdown in the management of proteome foldedness. Indeed, it is remarkable that under normal conditions cells can keep their proteome in a highly crowded and confined space without uncontrollable aggregation. Proteins pose a particular challenge relative to other classes of biomolecules because upon synthesis they must typically follow a complex folding pathway to reach their functional conformation (native state). Non-native conformations, including the unfolded nascent chain, are highly prone to aberrant interactions, leading to aggregation. Here we review recent advances in knowledge of proteostasis, approaches to monitor proteostasis and the impact that protein aggregation has on biology. We also include discussion of the outstanding challenges. © 2017 IUBMB Life, 69(2):49-54, 2017.
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ItemNo Preview AvailableWalking the tightrope: proteostasis and neurodegenerative disease(WILEY, 2016-05-01)A characteristic of many neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and amyotrophic lateral sclerosis (ALS), is the aggregation of specific proteins into protein inclusions and/or plaques in degenerating brains. While much of the aggregated protein consists of disease specific proteins, such as amyloid-β, α-synuclein, or superoxide dismutase1 (SOD1), many other proteins are known to aggregate in these disorders. Although the role of protein aggregates in the pathogenesis of neurodegenerative diseases remains unknown, the ubiquitous association of misfolded and aggregated proteins indicates that significant dysfunction in protein homeostasis (proteostasis) occurs in these diseases. Proteostasis is the concept that the integrity of the proteome is in fine balance and requires proteins in a specific conformation, concentration, and location to be functional. In this review, we discuss the role of specific mechanisms, both inside and outside cells, which maintain proteostasis, including molecular chaperones, protein degradation pathways, and the active formation of inclusions, in neurodegenerative diseases associated with protein aggregation. A characteristic of many neurodegenerative diseases is the aggregation of specific proteins, which alone provides strong evidence that protein homeostasis is disrupted in these disease states. Proteostasis is the maintenance of the proteome in the correct conformation, concentration, and location by functional pathways such as molecular chaperones and protein degradation machinery. Here, we discuss the potential roles of quality control pathways, both inside and outside cells, in the loss of proteostasis during aging and disease.
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ItemSize analysis of polyglutamine protein aggregates using fluorescence detection in an analytical ultracentrifuge.(Springer Nature, 2013)Defining the aggregation process of proteins formed by poly-amino acid repeats in cells remains a challenging task due to a lack of robust techniques for their isolation and quantitation. Sedimentation velocity methodology using fluorescence detected analytical ultracentrifugation is one approach that can offer significant insight into aggregation formation and kinetics. While this technique has traditionally been used with purified proteins, it is now possible for substantial information to be collected with studies using cell lysates expressing a GFP-tagged protein of interest. In this chapter, we describe protocols for sample preparation and setting up the fluorescence detection system in an analytical ultracentrifuge to perform sedimentation velocity experiments on cell lysates containing aggregates formed by poly-amino acid repeat proteins.
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ItemPulse shape analysis (PulSA) to track protein translocalization in cells by flow cytometry: applications for polyglutamine aggregation.(Springer Nature, 2013)Pulse shape analysis (PulSA) is a flow cytometry-based method that can be used to study protein localization patterns in cells. Examples for its use include tracking the formation of inclusion bodies of polyglutamine-expanded proteins and other aggregating proteins. The method can also be used for phenomena relating to protein movements in cells such as translocation from the cytoplasm to the nucleus, trafficking from the plasma membrane to the Golgi, and stress granule formation. An attractive feature is its capacity to quantify these parameters in whole-cell populations very quickly and in high throughput. We describe the basic experimental details for performing PulSA using expression of GFP-tagged proteins, endogenous proteins labelled immunofluorescently, and organelle dyes.
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ItemNo Preview AvailableThe Allosteric Mechanism Induced by Protein Kinase A (PKA) Phosphorylation of Dematin (Band 4.9)(AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2013-03-22)Dematin (band 4.9) is an F-actin binding and bundling protein best known for its role within red blood cells, where it both stabilizes as well as attaches the spectrin/actin cytoskeleton to the erythrocytic membrane. Here, we investigate the structural consequences of phosphorylating serine 381, a covalent modification that turns off F-actin bundling activity. In contrast to the canonical doctrine, in which phosphorylation of an intrinsically disordered region/protein confers affinity for another domain/protein, we found the converse to be true of dematin: phosphorylation of the well folded C-terminal villin-type headpiece confers affinity for its intrinsically disordered N-terminal core domain. We employed analytical ultracentrifugation to demonstrate that dematin is monomeric, in contrast to the prevailing view that it is trimeric. Next, using a series of truncation mutants, we verified that dematin has two F-actin binding sites, one in the core domain and the other in the headpiece domain. Although the phosphorylation-mimicking mutant, S381E, was incapable of bundling microfilaments, it retains the ability to bind F-actin. We found that a phosphorylation-mimicking mutant, S381E, eliminated the ability to bundle, but not bind F-actin filaments. Lastly, we show that the S381E point mutant caused the headpiece domain to associate with the core domain, leading us to the mechanism for cAMP-dependent kinase control of dematin's F-actin bundling activity: when unphosphorylated, dematin's two F-actin binding domains move independent of one another permitting them to bind different F-actin filaments. Phosphorylation causes these two domains to associate, forming a compact structure, and sterically eliminating one of these F-actin binding sites.