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1.
J Biol Chem ; 294(9): 3065-3080, 2019 03 01.
Article in English | MEDLINE | ID: mdl-30598510

ABSTRACT

Betaglycan (BG) is a membrane-bound co-receptor of the TGF-ß family that selectively binds transforming growth factor-ß (TGF-ß) isoforms and inhibin A (InhA) to enable temporal-spatial patterns of signaling essential for their functions in vivo Here, using NMR titrations of methyl-labeled TGF-ß2 with BG's C-terminal binding domain, BGZP-C, and surface plasmon resonance binding measurements with TGF-ß2 variants, we found that the BGZP-C-binding site on TGF-ß2 is located on the inner surface of its extended finger region. Included in this binding site are Ile-92, Lys-97, and Glu-99, which are entirely or mostly specific to the TGF-ß isoforms and the InhA α-subunit, but they are unconserved in other TGF-ß family growth factors (GFs). In accord with the proposed specificity-determining role of these residues, BG bound bone morphogenetic protein 2 (BMP-2) weakly or not at all, and TGF-ß2 variants with the corresponding residues from BMP-2 bound BGZP-C more weakly than corresponding alanine variants. The BGZP-C-binding site on InhA previously was reported to be located on the outside of the extended finger region, yet at the same time to include Ser-112 and Lys-119, homologous to TGF-ß2 Ile-92 and Lys-97, on the inside of the fingers. Therefore, it is likely that both TGF-ß2 and InhA bind BGZP-C through a site on the inside of their extended finger regions. Overall, these results identify the BGZP-C-binding site on TGF-ß2 and shed light on the specificity of BG for select TGF-ß-type GFs and the mechanisms by which BG influences their signaling.


Subject(s)
Inhibins/metabolism , Proteoglycans/chemistry , Proteoglycans/metabolism , Receptors, Transforming Growth Factor beta/chemistry , Receptors, Transforming Growth Factor beta/metabolism , Transforming Growth Factor beta2/chemistry , Transforming Growth Factor beta2/metabolism , Amino Acid Sequence , Animals , Binding Sites , Humans , Hydrogen-Ion Concentration , Mice , Models, Molecular , Protein Binding , Protein Domains , Protein Structure, Secondary , Rats , Substrate Specificity
2.
J Biol Chem ; 294(4): 1189-1201, 2019 01 25.
Article in English | MEDLINE | ID: mdl-30514764

ABSTRACT

Phospholipase B-mediated hydrolysis of phosphatidylcholine (PC) results in the formation of free fatty acids and glycerophosphocholine (GPC) in the yeast Saccharomyces cerevisiae GPC can be reacylated by the glycerophosphocholine acyltransferase Gpc1, which produces lysophosphatidylcholine (LPC), and LPC can be converted to PC by the lysophospholipid acyltransferase Ale1. Here, we further characterized the regulation and function of this distinct PC deacylation/reacylation pathway in yeast. Through in vitro and in vivo experiments, we show that Gpc1 and Ale1 are the major cellular GPC and LPC acyltransferases, respectively. Importantly, we report that Gpc1 activity affects the PC species profile. Loss of Gpc1 decreased the levels of monounsaturated PC species and increased those of diunsaturated PC species, whereas Gpc1 overexpression had the opposite effects. Of note, Gpc1 loss did not significantly affect phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine profiles. Our results indicate that Gpc1 is involved in postsynthetic PC remodeling that produces more saturated PC species. qRT-PCR analyses revealed that GPC1 mRNA abundance is regulated coordinately with PC biosynthetic pathways. Inositol availability, which regulates several phospholipid biosynthetic genes, down-regulated GPC1 expression at the mRNA and protein levels and, as expected, decreased levels of monounsaturated PC species. Finally, loss of GPC1 decreased stationary phase viability in inositol-free medium. These results indicate that Gpc1 is part of a postsynthetic PC deacylation/reacylation remodeling pathway (PC-DRP) that alters the PC species profile, is regulated in coordination with other major lipid biosynthetic pathways, and affects yeast growth.


Subject(s)
Acyltransferases/metabolism , Glycerylphosphorylcholine/metabolism , Phosphatidylcholines/chemistry , Phosphatidylcholines/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Acylation , Acyltransferases/chemistry , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/chemistry
3.
J Am Chem Soc ; 140(44): 14576-14580, 2018 11 07.
Article in English | MEDLINE | ID: mdl-30339373

ABSTRACT

A pathological hallmark of Huntington's disease (HD) is the formation of neuronal protein deposits containing mutant huntingtin fragments with expanded polyglutamine (polyQ) domains. Prior studies have shown the strengths of solid-state NMR (ssNMR) to probe the atomic structure of such aggregates, but have required in vitro isotopic labeling. Herein, we present an approach for the structural fingerprinting of fibrils through ssNMR at natural isotopic abundance (NA). These methods will enable the spectroscopic fingerprinting of unlabeled (e.g., ex vivo) protein aggregates and the extraction of valuable new long-range 13C-13C distance constraints.


Subject(s)
Huntingtin Protein/chemistry , Nuclear Magnetic Resonance, Biomolecular , Carbon Isotopes , Humans , Nitrogen Isotopes , Particle Size , Protein Aggregates , Protein Conformation
4.
J Biol Chem ; 293(4): 1353-1362, 2018 01 26.
Article in English | MEDLINE | ID: mdl-29217771

ABSTRACT

It has been long assumed that post-mitotic neurons only utilize the error-prone non-homologous end-joining pathway to repair double-strand breaks (DSBs) associated with oxidative damage to DNA, given the inability of non-replicating neuronal DNA to utilize a sister chromatid template in the less error-prone homologous recombination (HR) repair pathway. However, we and others have found recently that active transcription triggers a replication-independent recombinational repair mechanism in G0/G1 phase of the cell cycle. Here we observed that the HR repair protein RAD52 is recruited to sites of DNA DSBs in terminally differentiated, post-mitotic neurons. This recruitment is dependent on the presence of a nascent mRNA generated during active transcription, providing evidence that an RNA-templated HR repair mechanism exists in non-dividing, terminally differentiated neurons. This recruitment of RAD52 in neurons is decreased by transcription inhibition. Importantly, we found that high concentrations of amyloid ß, a toxic protein associated with Alzheimer's disease, inhibits the expression and DNA damage response of RAD52, potentially leading to a defect in the error-free, RNA-templated HR repair mechanism. This study shows a novel RNA-dependent repair mechanism of DSBs in post-mitotic neurons and demonstrates that defects in this pathway may contribute to neuronal genomic instability and consequent neurodegenerative phenotypes such as those seen in Alzheimer's disease.


Subject(s)
DNA Breaks, Double-Stranded , Mitosis/physiology , Neurons/metabolism , RNA/metabolism , Rad52 DNA Repair and Recombination Protein/metabolism , Recombination, Genetic/physiology , Animals , G1 Phase/physiology , Neurons/cytology , RNA/genetics , Rad52 DNA Repair and Recombination Protein/genetics , Rats , Resting Phase, Cell Cycle/physiology
5.
Nat Commun ; 8(1): 1741, 2017 11 23.
Article in English | MEDLINE | ID: mdl-29170498

ABSTRACT

Helminth parasites defy immune exclusion through sophisticated evasion mechanisms, including activation of host immunosuppressive regulatory T (Treg) cells. The mouse parasite Heligmosomoides polygyrus can expand the host Treg population by secreting products that activate TGF-ß signalling, but the identity of the active molecule is unknown. Here we identify an H. polygyrus TGF-ß mimic (Hp-TGM) that replicates the biological and functional properties of TGF-ß, including binding to mammalian TGF-ß receptors and inducing mouse and human Foxp3+ Treg cells. Hp-TGM has no homology with mammalian TGF-ß or other members of the TGF-ß family, but is a member of the complement control protein superfamily. Thus, our data indicate that through convergent evolution, the parasite has acquired a protein with cytokine-like function that is able to exploit an endogenous pathway of immunoregulation in the host.


Subject(s)
Molecular Mimicry/immunology , Nematospiroides dubius/immunology , Nematospiroides dubius/pathogenicity , T-Lymphocytes, Regulatory/immunology , Transforming Growth Factor beta/metabolism , Amino Acid Sequence , Animals , Antigens, Helminth/chemistry , Antigens, Helminth/genetics , Antigens, Helminth/immunology , Female , Helminth Proteins/chemistry , Helminth Proteins/genetics , Helminth Proteins/immunology , Host-Pathogen Interactions/genetics , Host-Pathogen Interactions/immunology , Humans , Immune Evasion/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic , Molecular Mimicry/genetics , Nematospiroides dubius/genetics , Protein Binding , Protein Domains , Receptors, Transforming Growth Factor beta/metabolism , Strongylida Infections/immunology , Strongylida Infections/parasitology
6.
Neuron ; 96(2): 355-372.e6, 2017 Oct 11.
Article in English | MEDLINE | ID: mdl-29024660

ABSTRACT

Compelling evidence links amyloid beta (Aß) peptide accumulation in the brains of Alzheimer's disease (AD) patients with the emergence of learning and memory deficits, yet a clear understanding of the events that drive this synaptic pathology are lacking. We present evidence that neurons exposed to Aß are unable to form new synapses, resulting in learning deficits in vivo. We demonstrate the Nogo receptor family (NgR1-3) acts as Aß receptors mediating an inhibition of synapse assembly, plasticity, and learning. Live imaging studies reveal Aß activates NgRs on the dendritic shaft of neurons, triggering an inhibition of calcium signaling. We define T-type calcium channels as a target of Aß-NgR signaling, mediating Aß's inhibitory effects on calcium, synapse assembly, plasticity, and learning. These studies highlight deficits in new synapse assembly as a potential initiator of cognitive pathology in AD, and pinpoint calcium dysregulation mediated by NgRs and T-type channels as key components. VIDEO ABSTRACT.


Subject(s)
Amyloid beta-Peptides/pharmacology , Calcium Channel Blockers/pharmacology , Calcium Channels, T-Type/physiology , Calcium Signaling/physiology , Nogo Receptors/physiology , Peptide Fragments/pharmacology , Synapses/physiology , Animals , CHO Cells , Calcium Signaling/drug effects , Cells, Cultured , Cricetinae , Cricetulus , Female , HEK293 Cells , Humans , Male , Mice , Mice, Knockout , Mice, Transgenic , Organ Culture Techniques , Rats , Rats, Long-Evans , Synapses/drug effects
8.
Nat Commun ; 8: 15462, 2017 05 24.
Article in English | MEDLINE | ID: mdl-28537272

ABSTRACT

Polyglutamine expansion in the huntingtin protein is the primary genetic cause of Huntington's disease (HD). Fragments coinciding with mutant huntingtin exon1 aggregate in vivo and induce HD-like pathology in mouse models. The resulting aggregates can have different structures that affect their biochemical behaviour and cytotoxic activity. Here we report our studies of the structure and functional characteristics of multiple mutant htt exon1 fibrils by complementary techniques, including infrared and solid-state NMR spectroscopies. Magic-angle-spinning NMR reveals that fibrillar exon1 has a partly mobile α-helix in its aggregation-accelerating N terminus, and semi-rigid polyproline II helices in the proline-rich flanking domain (PRD). The polyglutamine-proximal portions of these domains are immobilized and clustered, limiting access to aggregation-modulating antibodies. The polymorphic fibrils differ in their flanking domains rather than the polyglutamine amyloid structure. They are effective at seeding polyglutamine aggregation and exhibit cytotoxic effects when applied to neuronal cells.


Subject(s)
Amyloid/chemistry , Huntingtin Protein/genetics , Huntington Disease/genetics , Peptides/chemistry , Protein Aggregation, Pathological/genetics , Amyloid/genetics , Amyloid/metabolism , Amyloid/toxicity , Animals , Cell Line , Exons/genetics , Humans , Huntingtin Protein/chemistry , Huntingtin Protein/metabolism , Huntingtin Protein/toxicity , Huntington Disease/pathology , Magnetic Resonance Spectroscopy , Mice , Microscopy, Electron, Transmission , Mutation , Neurons , Peptides/genetics , Peptides/metabolism , Peptides/toxicity , Protein Aggregation, Pathological/pathology , Protein Structure, Secondary/genetics
9.
J Biol Chem ; 292(17): 7173-7188, 2017 04 28.
Article in English | MEDLINE | ID: mdl-28228478

ABSTRACT

The transforming growth factor ß isoforms, TGF-ß1, -ß2, and -ß3, are small secreted homodimeric signaling proteins with essential roles in regulating the adaptive immune system and maintaining the extracellular matrix. However, dysregulation of the TGF-ß pathway is responsible for promoting the progression of several human diseases, including cancer and fibrosis. Despite the known importance of TGF-ßs in promoting disease progression, no inhibitors have been approved for use in humans. Herein, we describe an engineered TGF-ß monomer, lacking the heel helix, a structural motif essential for binding the TGF-ß type I receptor (TßRI) but dispensable for binding the other receptor required for TGF-ß signaling, the TGF-ß type II receptor (TßRII), as an alternative therapeutic modality for blocking TGF-ß signaling in humans. As shown through binding studies and crystallography, the engineered monomer retained the same overall structure of native TGF-ß monomers and bound TßRII in an identical manner. Cell-based luciferase assays showed that the engineered monomer functioned as a dominant negative to inhibit TGF-ß signaling with a Ki of 20-70 nm Investigation of the mechanism showed that the high affinity of the engineered monomer for TßRII, coupled with its reduced ability to non-covalently dimerize and its inability to bind and recruit TßRI, enabled it to bind endogenous TßRII but prevented it from binding and recruiting TßRI to form a signaling complex. Such engineered monomers provide a new avenue to probe and manipulate TGF-ß signaling and may inform similar modifications of other TGF-ß family members.


Subject(s)
Protein Engineering/methods , Protein Serine-Threonine Kinases/antagonists & inhibitors , Receptors, Transforming Growth Factor beta/antagonists & inhibitors , Signal Transduction , Transforming Growth Factor beta/chemistry , Amino Acid Motifs , Animals , Disease Progression , Extracellular Matrix/metabolism , Fluorescence Resonance Energy Transfer , HEK293 Cells , Humans , Kinetics , Mice , Protein Binding , Protein Folding , Protein Isoforms , Protein Multimerization , Protein Serine-Threonine Kinases/metabolism , Receptor, Transforming Growth Factor-beta Type I , Receptors, Transforming Growth Factor beta/metabolism , Solubility , Surface Plasmon Resonance , Transforming Growth Factor beta/metabolism , Ultracentrifugation
10.
J Alzheimers Dis ; 56(3): 1075-1085, 2017.
Article in English | MEDLINE | ID: mdl-28106559

ABSTRACT

ATP-binding cassette transporter A1 (ABCA1) mediates cholesterol efflux to lipid-free apolipoproteins and regulates the generation of high density lipoproteins. Previously, we have shown that lack of Abca1 significantly increases amyloid deposition and cognitive deficits in Alzheimer's disease model mice expressing human amyloid-ß protein precursor (APP). The goal of this study was to determine if ABCA1 plays a role in memory deficits caused by amyloid-ß (Aß) oligomers and examine neurite architecture of pyramidal hippocampal neurons. Our results confirm previous findings that Abca1 deficiency significantly impairs spatial memory acquisition and retention in the Morris water maze and long-term memory in novel object recognition of APP transgenic mice at a stage of early amyloid pathology. Neither test demonstrated a significant difference between Abca1ko and wild-type (WT) mice. We also examined the effect of intra-hippocampal infused Aß oligomers on cognitive performance of Abca1ko mice, compared to control infusion of scrambled Aß peptide. Age-matched WT mice undergoing the same infusions were also used as controls. In this model system, we found a statistically significant difference between WT and Abca1ko mice infused with scrambled Aß, suggesting that Abca1ko mice are vulnerable to the effect of mild stresses. Moreover, examination of neurite architecture in the hippocampi revealed a significant decrease in neurite length, number of neurite segments, and branches in Abca1ko mice when compared to WT mice. We conclude that mice lacking ABCA1 have basal cognitive deficits that prevent them from coping with additional stressors, which is in part due to impairment of neurite morphology in the hippocampus.


Subject(s)
ATP Binding Cassette Transporter 1/deficiency , Cognition Disorders/metabolism , Cognition Disorders/pathology , Dendrites/metabolism , Dendrites/pathology , ATP Binding Cassette Transporter 1/genetics , Amyloid beta-Peptides/metabolism , Amyloid beta-Protein Precursor/genetics , Amyloid beta-Protein Precursor/metabolism , Animals , Cell Size , Female , Hippocampus/metabolism , Hippocampus/pathology , Humans , Male , Maze Learning/physiology , Mice, Inbred C57BL , Mice, Transgenic , Presenilin-1/genetics , Presenilin-1/metabolism , Recognition, Psychology/physiology , Spatial Memory/physiology
11.
J Mol Biol ; 429(2): 308-323, 2017 01 20.
Article in English | MEDLINE | ID: mdl-27986569

ABSTRACT

Candidates for the toxic molecular species in the expanded polyglutamine (polyQ) repeat diseases range from various types of aggregates to "misfolded" monomers. One way to vet these candidates is to develop mutants that restrict conformational landscapes. Previously, we inserted two self-complementary ß-hairpin enhancing motifs into a short polyQ sequence to generate a mutant, here called "ßHP," that exhibits greatly improved amyloid nucleation without measurably enhancing ß-structure in the monomer ensemble. We extend these studies here by introducing single-backbone H-bond impairing modifications αN-methyl Gln or l-Pro at key positions within ßHP. Modifications predicted to allow formation of a fully H-bonded ß-hairpin at the fibril edge while interfering with H-bonding to the next incoming monomer exhibit poor amyloid formation and act as potent inhibitors in trans of simple polyQ peptide aggregation. In contrast, a modification that disrupts intra-ß-hairpin H-bonding within ßHP, while also aggregating poorly, is ineffective at inhibiting amyloid formation in trans. The inhibitors constitute a dynamic version of the edge-protection negative design strategy used in protein evolution to limit unwanted protein aggregation. Our data support a model in which polyQ peptides containing strong ß-hairpin encouraging motifs only rarely form ß-hairpin conformations in the monomer ensemble, but nonetheless take on such conformations at key steps during amyloid formation. The results provide insights into polyQ solution structure and fibril formation while also suggesting an approach to the design of inhibitors of polyQ amyloid growth that focuses on conformational requirements for fibril and nucleus elongation.


Subject(s)
Amyloid beta-Peptides/chemistry , Peptides/chemistry , Protein Engineering , Amino Acid Sequence , Hydrogen-Ion Concentration , Magnetic Resonance Spectroscopy , Peptides/antagonists & inhibitors , Protein Interaction Domains and Motifs , Protein Structure, Secondary , Reproducibility of Results
12.
Oncotarget ; 7(52): 86087-86102, 2016 Dec 27.
Article in English | MEDLINE | ID: mdl-27863384

ABSTRACT

The effects of transforming growth factor beta (TGF-ß) signaling on prostate tumorigenesis has been shown to be strongly dependent on the stage of development, with TGF-ß functioning as a tumor suppressor in early stages of disease and as a promoter in later stages. To study in further detail the paradoxical tumor-suppressive and tumor-promoting roles of the TGF-ß pathway, we investigated the effect of systemic treatment with a TGF-ß inhibitor on early stages of prostate tumorigenesis. To ensure effective inhibition, we developed and employed a novel trivalent TGF-ß receptor trap, RER, comprised of domains derived from the TGF-ß type II and type III receptors. This trap was shown to completely block TßRII binding, to antagonize TGF-ß1 and TGF-ß3 signaling in cultured epithelial cells at low picomolar concentrations, and it showed equal or better anti-TGF-ß activities than a pan TGF-ß neutralizing antibody and a TGF-ß receptor I kinase inhibitor in various prostate cancer cell lines. Systemic administration of RER inhibited prostate tumor cell proliferation as indicated by reduced Ki67 positive cells and invasion potential of tumor cells in high grade prostatic intraepithelial neoplasia (PIN) lesions in the prostate glands of Pten conditional null mice. These results provide evidence that TGF-ß acts as a promoter rather than a suppressor in the relatively early stages of this spontaneous prostate tumorigenesis model. Thus, inhibition of TGF-ß signaling in early stages of prostate cancer may be a novel therapeutic strategy to inhibit the progression as well as the metastatic potential in patients with prostate cancer.


Subject(s)
PTEN Phosphohydrolase/physiology , Prostate/pathology , Prostatic Neoplasms/prevention & control , Receptors, Transforming Growth Factor beta/antagonists & inhibitors , Animals , Carcinogenesis , Cell Line, Tumor , Cell Proliferation , Humans , Male , Mice , Neoplasm Invasiveness , Neoplasm Staging , Phosphorylation , Prostatic Neoplasms/pathology , Proto-Oncogene Proteins c-akt/physiology , Signal Transduction/physiology , Smad Proteins/metabolism
13.
Nat Commun ; 7: 12419, 2016 08 22.
Article in English | MEDLINE | ID: mdl-27546208

ABSTRACT

Since early oligomeric intermediates in amyloid assembly are often transient and difficult to distinguish, characterize and quantify, the mechanistic basis of the initiation of spontaneous amyloid growth is often opaque. We describe here an approach to the analysis of the Aß aggregation mechanism that uses Aß-polyglutamine hybrid peptides designed to retard amyloid maturation and an adjusted thioflavin intensity scale that reveals structural features of aggregation intermediates. The results support an aggregation initiation mechanism for Aß-polyQ hybrids, and by extension for full-length Aß peptides, in which a modular Aß C-terminal segment mediates rapid, non-nucleated formation of α-helical oligomers. The resulting high local concentration of tethered amyloidogenic segments within these α-oligomers facilitates transition to a ß-oligomer population that, via further remodelling and/or elongation steps, ultimately generates mature amyloid. Consistent with this mechanism, an engineered Aß C-terminal fragment delays aggregation onset by Aß-polyglutamine peptides and redirects assembly of Aß42 fibrils.


Subject(s)
Alzheimer Disease/pathology , Amyloid beta-Peptides/metabolism , Peptide Fragments/metabolism , Peptides/metabolism , Protein Aggregation, Pathological/pathology , Protein Multimerization , Amyloid beta-Peptides/chemistry , Amyloid beta-Peptides/genetics , Benzothiazoles , Circular Dichroism , Fluorescent Dyes/chemistry , Humans , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptides/chemistry , Peptides/genetics , Protein Engineering , Protein Structure, Secondary , Spectrum Analysis/methods , Thiazoles/chemistry , Time Factors
14.
PLoS One ; 11(6): e0155747, 2016.
Article in English | MEDLINE | ID: mdl-27271685

ABSTRACT

Expansion of the polyglutamine (polyQ) track of the Huntingtin (HTT) protein above 36 is associated with a sharply enhanced risk of Huntington's disease (HD). Although there is general agreement that HTT toxicity resides primarily in N-terminal fragments such as the HTT exon1 protein, there is no consensus on the nature of the physical states of HTT exon1 that are induced by polyQ expansion, nor on which of these states might be responsible for toxicity. One hypothesis is that polyQ expansion induces an alternative, toxic conformation in the HTT exon1 monomer. Alternative hypotheses posit that the toxic species is one of several possible aggregated states. Defining the nature of the toxic species is particularly challenging because of facile interconversion between physical states as well as challenges to identifying these states, especially in vivo. Here we describe the use of fluorescence correlation spectroscopy (FCS) to characterize the detailed time and repeat length dependent self-association of HTT exon1-like fragments both with chemically synthesized peptides in vitro and with cell-produced proteins in extracts and in living cells. We find that, in vitro, mutant HTT exon1 peptides engage in polyQ repeat length dependent dimer and tetramer formation, followed by time dependent formation of diffusible spherical and fibrillar oligomers and finally by larger, sedimentable amyloid fibrils. For expanded polyQ HTT exon1 expressed in PC12 cells, monomers are absent, with tetramers being the smallest molecular form detected, followed in the incubation time course by small, diffusible aggregates at 6-9 hours and larger, sedimentable aggregates that begin to build up at 12 hrs. In these cell cultures, significant nuclear DNA damage appears by 6 hours, followed at later times by caspase 3 induction, mitochondrial dysfunction, and cell death. Our data thus defines limits on the sizes and concentrations of different physical states of HTT exon1 along the reaction profile in the context of emerging cellular distress. The data provide some new candidates for the toxic species and some new reservations about more well-established candidates. Compared to other known markers of HTT toxicity, nuclear DNA damage appears to be a relatively early pathological event.


Subject(s)
Huntingtin Protein/chemistry , Huntingtin Protein/genetics , Mutant Proteins/chemistry , Mutant Proteins/genetics , Protein Multimerization , Amyloid/chemistry , Amyloid/genetics , Amyloid/metabolism , Animals , Cell Survival/genetics , DNA Damage/genetics , Huntingtin Protein/metabolism , Mutant Proteins/metabolism , Mutation/physiology , PC12 Cells , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Peptides/chemistry , Peptides/genetics , Peptides/metabolism , Protein Folding , Rats
15.
J Mol Biol ; 428(2 Pt A): 274-291, 2016 Jan 29.
Article in English | MEDLINE | ID: mdl-26122432

ABSTRACT

Aß43, a product of the proteolysis of the amyloid precursor protein APP, is related to Aß42 by an additional Thr residue at the C-terminus. Aß43 is typically generated at low levels compared with the predominant Aß42 and Aß40 forms, but it has been suggested that this longer peptide might have an impact on amyloid-ß aggregation and Alzheimer's disease that is out of proportion to its brain content. Here, we report that both Aß42 and Aß43 spontaneously aggregate into mature amyloid fibrils via sequential appearance of the same series of oligomeric and protofibrillar intermediates, the earliest of which appears to lack ß-structure. In spite of the additional ß-branched amino acid at the C-terminus, Aß43 fibrils have fewer strong backbone H-bonds than Aß42 fibrils, some of which are lost at the C-terminus. In contrast to previous reports, we found that Aß43 spontaneously aggregates more slowly than Aß42. In addition, Aß43 fibrils are very inefficient at seeding Aß42 amyloid formation, even though Aß42 fibrils efficiently seed amyloid formation by Aß43 monomers. Finally, mixtures of Aß42 and Aß43 aggregate more slowly than Aß42 alone. Both in this Aß42/Aß43 co-aggregation reaction and in cross-seeding by Aß42 fibrils, the structure of the Aß43 in the product fibrils is influenced by the presence of Aß42. The results provide new details of amyloid structure and assembly pathways, an example of structural plasticity in prion-like replication, and data showing that low levels of Aß43 in the brain are unlikely to favorably impact the aggregation of Aß42.


Subject(s)
Amyloid beta-Peptides/chemistry , Amyloid beta-Peptides/metabolism , Protein Aggregation, Pathological , Protein Denaturation , Protein Multimerization , Threonine/metabolism
16.
Biophys J ; 109(9): 1873-84, 2015 Nov 03.
Article in English | MEDLINE | ID: mdl-26536264

ABSTRACT

The cellular process of intrinsic apoptosis relies on the peroxidation of mitochondrial lipids as a critical molecular signal. Lipid peroxidation is connected to increases in mitochondrial reactive oxygen species, but there is also a required role for mitochondrial cytochrome c (cyt-c). In apoptotic mitochondria, cyt-c gains a new function as a lipid peroxidase that catalyzes the reactive oxygen species-mediated chemical modification of the mitochondrial lipid cardiolipin (CL). This peroxidase activity is caused by a conformational change in the protein, resulting from interactions between cyt-c and CL. The nature of the conformational change and how it causes this gain-of-function remain uncertain. Via a combination of functional, structural, and biophysical experiments we investigate the structure and peroxidase activity of cyt-c in its membrane-bound state. We reconstituted cyt-c with CL-containing lipid vesicles, and determined the increase in peroxidase activity resulting from membrane binding. We combined these assays of CL-induced proapoptotic activity with structural and dynamic studies of the membrane-bound protein via solid-state NMR and optical spectroscopy. Multidimensional magic angle spinning (MAS) solid-state NMR of uniformly (13)C,(15)N-labeled protein was used to detect site-specific conformational changes in oxidized and reduced horse heart cyt-c bound to CL-containing lipid bilayers. MAS NMR and Fourier transform infrared measurements show that the peripherally membrane-bound cyt-c experiences significant dynamics, but also retains most or all of its secondary structure. Moreover, in two-dimensional and three-dimensional MAS NMR spectra the CL-bound cyt-c displays a spectral resolution, and thus structural homogeneity, that is inconsistent with extensive membrane-induced unfolding. Cyt-c is found to interact primarily with the membrane interface, without significantly disrupting the lipid bilayer. Thus, membrane binding results in cyt-c gaining the increased peroxidase activity that represents its pivotal proapoptotic function, but we do not observe evidence for large-scale unfolding or penetration into the membrane core.


Subject(s)
Apoptosis/physiology , Cardiolipins/metabolism , Cytochromes c/metabolism , Mitochondria/metabolism , Peroxidase/metabolism , Carbon-13 Magnetic Resonance Spectroscopy , Escherichia coli , Lipid Bilayers/chemistry , Nuclear Magnetic Resonance, Biomolecular , Phosphatidylcholines/chemistry , Protein Conformation , Spectroscopy, Fourier Transform Infrared , Spectrum Analysis
17.
Biochemistry ; 53(24): 3897-907, 2014 Jun 24.
Article in English | MEDLINE | ID: mdl-24921664

ABSTRACT

Repeat length disease thresholds vary among the 10 expanded polyglutamine (polyQ) repeat diseases, from about 20 to about 50 glutamine residues. The unique amino acid sequences flanking the polyQ segment are thought to contribute to these repeat length thresholds. The specific portions of the flanking sequences that modulate polyQ properties are not always clear, however. This ambiguity may be important in Huntington's disease (HD), for example, where in vitro studies of aggregation mechanisms have led to distinctly different mechanistic models. Most in vitro studies of the aggregation of the huntingtin (HTT) exon1 fragment implicated in the HD mechanism have been conducted on inexact molecules that are imprecise either on the N-terminus (recombinantly produced peptides) or on the C-terminus (chemically synthesized peptides). In this paper, we investigate the aggregation properties of chemically synthesized HTT exon1 peptides that are full-length and complete, containing both normal and expanded polyQ repeat lengths, and compare the results directly to previously investigated molecules containing truncated C-termini. The results on the full-length peptides are consistent with a two-step aggregation mechanism originally developed based on studies of the C-terminally truncated analogues. Thus, we observe relatively rapid formation of spherical oligomers containing from 100 to 600 HTT exon1 molecules and intermediate formation of short protofibril-like structures containing from 500 to 2600 molecules. In contrast to this relatively rapid assembly, mature HTT exon1 amyloid requires about one month to dissociate in vitro, which is similar to the time required for neuronal HTT exon1 aggregates to disappear in vivo after HTT production is discontinued.


Subject(s)
Nerve Tissue Proteins/chemistry , Protein Structure, Quaternary , Exons , Humans , Huntingtin Protein , Huntington Disease/genetics , Kinetics , Nerve Tissue Proteins/chemical synthesis , Peptides/chemistry
18.
J Alzheimers Dis ; 41(2): 535-49, 2014.
Article in English | MEDLINE | ID: mdl-24643138

ABSTRACT

Passive amyloid-ß (Aß) vaccination has shown significant effects on amyloid pathology in pre-depositing amyloid-ß protein precursor (AßPP) mice but the results in older mice are inconsistent. A therapeutic effect of LXR and RXR agonists consisting of improved memory deficits and Aß pathology has been demonstrated in different Alzheimer's disease (AD) mouse models. Here, we report the effect of a combination of N-terminal Aß antibody and synthetic LXR agonist T0901317 (T0) on AD-like phenotype of APP23 mice. To examine the therapeutic potential of this combination, the treatment of mice started at 11 months of age, when amyloid phenotype in this model is fully developed, and continued for 50 days. We show that Aß immunization with or without LXR agonist restored the performance of APP23 transgenic mice in two behavior paradigms without affecting the existing amyloid plaques. Importantly, we did not observe an increase of brain microhemorrhage which is considered a significant side effect of Aß vaccination. Target engagement was confirmed by increased Abca1 and ApoE protein level as well as increased ApoE lipidation in soluble brain extract. In interstitial fluid obtained by microdialysis, we demonstrate that immunization and T0 significantly reduced Aß levels, indicating an increased Aß clearance. We found no interaction between the immunotherapy and T0, suggesting no synergism, at least with these doses. The results of our study demonstrate that anti-Aß treatments can ameliorate cognitive deficits in AßPP mice with advanced AD-like phenotype in conjunction with a decrease of Aß in brain interstitium and increase of ApoE lipidation without affecting the existing amyloid plaques.


Subject(s)
Alzheimer Disease/therapy , Amyloid beta-Peptides/metabolism , Antibodies, Monoclonal/therapeutic use , Hydrocarbons, Fluorinated/therapeutic use , Immunization, Passive , Memory Disorders/therapy , Sulfonamides/therapeutic use , ATP Binding Cassette Transporter 1/metabolism , Amyloid beta-Protein Precursor/genetics , Amyloidogenic Proteins/immunology , Animals , Apolipoproteins E/metabolism , Brain/drug effects , Brain/pathology , Brain/physiopathology , Combined Modality Therapy , Conditioning, Psychological/drug effects , Conditioning, Psychological/physiology , Disease Models, Animal , Fear/drug effects , Fear/physiology , Female , Male , Maze Learning/drug effects , Maze Learning/physiology , Memory Disorders/pathology , Memory Disorders/physiopathology , Mice, Inbred C57BL , Mice, Transgenic , Nootropic Agents/therapeutic use , Random Allocation
19.
Biopolymers ; 102(2): 206-21, 2014 Mar.
Article in English | MEDLINE | ID: mdl-24488729

ABSTRACT

Many amyloidogenic peptides are highly hydrophobic, introducing significant challenges to obtaining high quality peptides by chemical synthesis. For example, while good yield and purity can be obtained in the solid-phase synthesis of the Alzheimer's plaque peptide Aß40, addition of a C-terminal Ile-Ala sequence to generate the more toxic Aß42 molecule creates a much more difficult synthesis resulting in low yields and purities. We describe here a new method that significantly improves the Fmoc solid-phase synthesis of Aß peptides. In our method, Lys residues are linked to the desired peptide's C-terminus through standard peptide bonds during the synthesis. These Lys residues are then removed post-purification using immobilized carboxypeptidase B (CPB). With this method we obtained both Aß42 and Aß46 of superior quality that, for Aß42, rivals that obtained by recombinant expression. Intriguingly, the method appears to provide independent beneficial effects on both the total synthetic yield and on purification yield and final purity. Reversible Lys addition with CPB removal should be a generally useful method for making hydrophobic peptides that is applicable to any sequence not ending in Arg or Lys. As expected from the additional hydrophobicity of Aß46, which is extended from the sequence Aß42 by a C-terminal Thr-Val-Ile-Val sequence, this peptide makes typical amyloid at rates significantly faster than for Aß42 or Aß40. The enhanced amyloidogenicity of Aß46 suggests that, even though it is present in relatively low amounts in the human brain, it could play a significant role in helping to initiate Aß amyloid formation.


Subject(s)
Amyloid beta-Peptides/chemical synthesis , Carboxypeptidase B/metabolism , Hydrophobic and Hydrophilic Interactions , Lysine/metabolism , Amino Acid Sequence , Amyloid beta-Peptides/chemistry , Amyloid beta-Peptides/isolation & purification , Amyloid beta-Peptides/ultrastructure , Chromatography, High Pressure Liquid , Chromatography, Reverse-Phase , Enzymes, Immobilized/metabolism , Kinetics , Molecular Sequence Data , Protein Aggregates , Spectrometry, Mass, Electrospray Ionization , Spectroscopy, Fourier Transform Infrared , Temperature
20.
Biopolymers ; 100(6): 780-9, 2013 Nov.
Article in English | MEDLINE | ID: mdl-23893755

ABSTRACT

The 17- amino acid N-terminal segment of the Huntingtin protein, htt(NT), grows into stable α-helix rich oligomeric aggregates when incubated under physiological conditions. We examined 15 scrambled sequence versions of an htt(NT) peptide for their stabilities against aggregation in aqueous solution at low micromolar concentration and physiological conditions. Surprisingly, given their derivation from a sequence that readily assembles into highly stable α-helical aggregates that fail to convert into ß-structure, we found that three of these scrambled peptides rapidly grow into amyloid-like fibrils, while two others also develop amyloid somewhat more slowly. The other 10 scrambled peptides do not detectibly form any aggregates after 100 h incubation under these conditions. We then analyzed these sequences using four previously described algorithms for predicting the tendencies of peptides to grow into amyloid or other ß-aggregates. We found that these algorithms-Zyggregator, Tango, Waltz, and Zipper-varied greatly in the number of sequences predicted to be amyloidogenic and in their abilities to correctly identify the amyloid forming members of this scrambled peptide collection. The results are discussed in the context of a review of the sequence and structural factors currently thought to be important in determining amyloid formation kinetics and thermodynamics.


Subject(s)
Amyloid , Protein Structure, Secondary , Algorithms , Amino Acid Sequence , Amyloid/chemistry , Peptides/chemistry , Surveys and Questionnaires
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