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1.
Curr Biol ; 33(23): 5160-5168.e7, 2023 12 04.
Article in English | MEDLINE | ID: mdl-37989309

ABSTRACT

Toxic cardiotonic steroids (CTSs) act as a defense mechanism in many firefly species (Lampyridae) by inhibiting a crucial enzyme called Na+,K+-ATPase (NKA). Although most fireflies produce these toxins internally, species of the genus Photuris acquire them from a surprising source: predation on other fireflies. The contrasting physiology of toxin exposure and sequestration between Photuris and other firefly genera suggests that distinct strategies may be required to prevent self-intoxication. Our study demonstrates that both Photuris and their firefly prey have evolved highly resistant NKAs. Using an evolutionary analysis of the specific target of CTS (ATPα) in fireflies and gene editing in Drosophila, we find that the initial steps toward resistance were shared among Photuris and other firefly lineages. However, the Photuris lineage subsequently underwent multiple rounds of gene duplication and neofunctionalization, resulting in the development of ATPα paralogs that are differentially expressed and exhibit increasing resistance to CTS. By contrast, other firefly species have maintained a single copy. Our results implicate gene duplication as a facilitator in the transition of Photuris to its distinct ecological role as a predator of toxic firefly prey.


Subject(s)
Fireflies , Predatory Behavior , Animals , Biological Evolution
2.
bioRxiv ; 2023 Oct 24.
Article in English | MEDLINE | ID: mdl-36945443

ABSTRACT

Toxic cardiotonic steroids (CTS) act as a defense mechanism in many firefly species (Lampyridae) by inhibiting a crucial enzyme called Na+,K+-ATPase (NKA). While most fireflies produce these toxins internally, species of the genus Photuris acquire them from a surprising source: predation on other fireflies. The contrasting physiology of toxin exposure and sequestration between Photuris and other firefly genera suggests that distinct strategies may be required to prevent self-intoxication. Our study demonstrates that both Photuris and their firefly prey have evolved highly-resistant NKAs. Using an evolutionary analysis of the specific target of CTS (ATPα) in fireflies, and gene-editing in Drosophila, we find that the initial steps towards resistance were shared among Photuris and other firefly lineages. However, the Photuris lineage subsequently underwent multiple rounds of gene duplication and neofunctionalization, resulting in the development of ATPα paralogs that are differentially expressed and exhibit increasing resistance to CTS. In contrast, other firefly species have maintained a single copy. Our results implicate gene duplication as a facilitator in the transition of Photuris to its distinct ecological role as predator of toxic firefly prey.

3.
J Pers Med ; 11(11)2021 Oct 20.
Article in English | MEDLINE | ID: mdl-34834403

ABSTRACT

Pharmacogenomic (PGx) evidence for selective serotonin reuptake inhibitors (SSRIs) continues to evolve. For sites offering testing, maintaining up-to-date interpretations and implementing new clinical decision support (CDS) driven by existing results creates practical and technical challenges. Vanderbilt University Medical Center initiated panel testing in 2010, added CYP2D6 testing in 2017, and released CDS for SSRIs in 2020. We systematically reinterpreted historic CYP2C19 and CYP2D6 genotypes to update phenotypes to current nomenclature and to launch provider CDS and patient-oriented content for SSRIs. Chart review was conducted to identify and recontact providers caring for patients with current SSRI therapy and new actionable recommendations. A total of 15,619 patients' PGx results were reprocessed. Of the non-deceased patients reprocessed, 21% (n = 3278) resulted in CYP2C19*1/*17 reinterpretations. Among 289 patients with an actionable recommendation and SSRI medication prescription, 31.8% (n = 92) did not necessitate contact of a clinician, while 43.2% (n = 125) resulted in clinician contacted, and for 25% (n = 72) no appropriate clinician was able to be identified. Maintenance of up-to-date interpretations and recommendations for PGx results over the lifetime of a patient requires continuous effort. Reprocessing is a key strategy for maintenance and expansion of PGx content to be periodically considered and implemented.

4.
Clin Pharmacol Ther ; 109(1): 101-115, 2021 01.
Article in English | MEDLINE | ID: mdl-33048353

ABSTRACT

Vanderbilt University Medical Center implemented pharmacogenomics (PGx) testing with the Pharmacogenomic Resource for Enhanced Decisions in Care and Treatment (PREDICT) initiative in 2010. This tutorial reviews the laboratory considerations, technical infrastructure, and programmatic support required to deliver panel-based PGx testing across a large health system with examples and experiences from the first decade of the PREDICT initiative. From the time of inception, automated clinical decision support (CDS) has been a critical capability for delivering PGx results to the point-of-care. Key features of the CDS include human-readable interpretations and clinical guidance that is anticipatory, actionable, and adaptable to changes in the scientific literature. Implementing CDS requires that structured results from the laboratory be encoded in standards-based messages that are securely ingested by electronic health records. Translating results to guidance also requires an informatics infrastructure with multiple components: (1) to manage the interpretation of raw genomic data to "star allele" results to expected phenotype, (2) to define the rules that associate a phenotype with recommended changes to clinical care, and (3) to manage and update the knowledge base. Knowledge base management is key to processing new results with the latest guidelines, and to ensure that historical genomic results can be reinterpreted with revised CDS. We recommend that these components be deployed with institutional authorization, programmatic support, and clinician education to govern the CDS content and policies around delivery.


Subject(s)
Decision Support Systems, Clinical/standards , Pharmacogenetics/methods , Pharmacogenetics/standards , Genomics/standards , Humans , Point-of-Care Systems/standards , Precision Medicine/methods , Precision Medicine/standards
5.
J Biol Chem ; 295(52): 17997-18009, 2020 12 25.
Article in English | MEDLINE | ID: mdl-33060204

ABSTRACT

The plasma membrane of a cell is characterized by an asymmetric distribution of lipid species across the exofacial and cytofacial aspects of the bilayer. Regulation of membrane asymmetry is a fundamental characteristic of membrane biology and is crucial for signal transduction, vesicle transport, and cell division. The type IV family of P-ATPases, or P4-ATPases, establishes membrane asymmetry by selection and transfer of a subset of membrane lipids from the lumenal or exofacial leaflet to the cytofacial aspect of the bilayer. It is unclear how P4-ATPases sort through the spectrum of membrane lipids to identify their desired substrate(s) and how the membrane environment modulates this activity. Therefore, we tested how the yeast plasma membrane P4-ATPase, Dnf2, responds to changes in membrane composition induced by perturbation of endogenous lipid biosynthetic pathways or exogenous application of lipid. The primary substrates of Dnf2 are glucosylceramide (GlcCer) and phosphatidylcholine (PC, or their lyso-lipid derivatives), and we find that these substrates compete with each other for transport. Acutely inhibiting sphingolipid synthesis using myriocin attenuates transport of exogenously applied GlcCer without perturbing PC transport. Deletion of genes controlling later steps of glycosphingolipid production also perturb GlcCer transport to a greater extent than PC transport. In contrast, perturbation of ergosterol biosynthesis reduces PC and GlcCer transport equivalently. Surprisingly, application of lipids that are poor transport substrates differentially affects PC and GlcCer transport by Dnf2, thus altering substrate preference. Our data indicate that Dnf2 exhibits exquisite sensitivity to the membrane composition, thus providing feedback onto the function of the P4-ATPases.


Subject(s)
ATP-Binding Cassette Transporters/metabolism , Adenosine Triphosphatases/metabolism , Cell Membrane/chemistry , Cell Membrane/metabolism , Lipid Metabolism , Membrane Lipids/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Sphingolipids/metabolism , ATP-Binding Cassette Transporters/genetics , Adenosine Triphosphatases/genetics , Biological Transport , Models, Molecular , Phospholipids/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae Proteins/genetics , Substrate Specificity
6.
Elife ; 82019 08 27.
Article in English | MEDLINE | ID: mdl-31453806

ABSTRACT

Predicting how species will respond to selection pressures requires understanding the factors that constrain their evolution. We use genome engineering of Drosophila to investigate constraints on the repeated evolution of unrelated herbivorous insects to toxic cardiac glycosides, which primarily occurs via a small subset of possible functionally-relevant substitutions to Na+,K+-ATPase. Surprisingly, we find that frequently observed adaptive substitutions at two sites, 111 and 122, are lethal when homozygous and adult heterozygotes exhibit dominant neural dysfunction. We identify a phylogenetically correlated substitution, A119S, that partially ameliorates the deleterious effects of substitutions at 111 and 122. Despite contributing little to cardiac glycoside-insensitivity in vitro, A119S, like substitutions at 111 and 122, substantially increases adult survivorship upon cardiac glycoside exposure. Our results demonstrate the importance of epistasis in constraining adaptive paths. Moreover, by revealing distinct effects of substitutions in vitro and in vivo, our results underscore the importance of evaluating the fitness of adaptive substitutions and their interactions in whole organisms.


Subject(s)
Adaptation, Biological , Cardiac Glycosides/pharmacology , Drosophila/drug effects , Drosophila/genetics , Epistasis, Genetic , Insecticide Resistance , Insecticides/pharmacology , Animals
7.
Biochim Biophys Acta Mol Basis Dis ; 1865(9): 2257-2266, 2019 09 01.
Article in English | MEDLINE | ID: mdl-31075491

ABSTRACT

Mutations in the gene triosephosphate isomerase (TPI) lead to a severe multisystem condition that is characterized by hemolytic anemia, a weakened immune system, and significant neurologic symptoms such as seizures, distal neuropathy, and intellectual disability. No effective therapy is available. Here we report a compound heterozygous patient with a novel TPI pathogenic variant (NM_000365.5:c.569G>A:p.(Arg189Gln)) in combination with the common (NM_000365.5:c.315G>C:p.(Glu104Asp)) allele. We characterized the novel variant by mutating the homologous Arg in Drosophila using a genomic engineering system, demonstrating that missense mutations at this position cause a strong loss of function. Compound heterozygote animals were generated and exhibit motor behavioural deficits and markedly reduced protein levels. Furthermore, examinations of the TPIArg189Gln/TPIGlu104Asp patient fibroblasts confirmed the reduction of TPI levels, suggesting that Arg189Gln may also affect the stability of the protein. The Arg189 residue participates in two salt bridges on the backside of the TPI enzyme dimer, and we reveal that a mutation at this position alters the coordination of the substrate-binding site and important catalytic residues. Collectively, these data reveal a new human pathogenic variant associated with TPI deficiency, identify the Arg189 salt bridge as critical for organizing the catalytic site of the TPI enzyme, and demonstrates that reduced TPI levels are associated with human TPI deficiency. These findings advance our understanding of the molecular pathogenesis of the disease, and suggest new therapeutic avenues for pre-clinical trials.


Subject(s)
Anemia, Hemolytic, Congenital Nonspherocytic/pathology , Carbohydrate Metabolism, Inborn Errors/pathology , Triose-Phosphate Isomerase/deficiency , Triose-Phosphate Isomerase/metabolism , Alleles , Amino Acid Sequence , Anemia, Hemolytic, Congenital Nonspherocytic/genetics , Animals , Base Sequence , Carbohydrate Metabolism, Inborn Errors/genetics , Catalytic Domain , Child, Preschool , Dimerization , Disease Models, Animal , Drosophila/metabolism , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Female , Fibroblasts/cytology , Fibroblasts/metabolism , Humans , Mutation, Missense , Pedigree , Protein Stability , Sequence Alignment , Triose-Phosphate Isomerase/genetics
8.
J Biol Chem ; 294(6): 1794-1806, 2019 02 08.
Article in English | MEDLINE | ID: mdl-30530492

ABSTRACT

Lipid transport is an essential process with manifest importance to human health and disease. Phospholipid flippases (P4-ATPases) transport lipids across the membrane bilayer and are involved in signal transduction, cell division, and vesicular transport. Mutations in flippase genes cause or contribute to a host of diseases, such as cholestasis, neurological deficits, immunological dysfunction, and metabolic disorders. Genome-wide association studies have shown that ATP10A and ATP10D variants are associated with an increased risk of diabetes, obesity, myocardial infarction, and atherosclerosis. Moreover, ATP10D SNPs are associated with elevated levels of glucosylceramide (GlcCer) in plasma from diverse European populations. Although sphingolipids strongly contribute to metabolic disease, little is known about how GlcCer is transported across cell membranes. Here, we identify a conserved clade of P4-ATPases from Saccharomyces cerevisiae (Dnf1, Dnf2), Schizosaccharomyces pombe (Dnf2), and Homo sapiens (ATP10A, ATP10D) that transport GlcCer bearing an sn2 acyl-linked fluorescent tag. Further, we establish structural determinants necessary for recognition of this sphingolipid substrate. Using enzyme chimeras and site-directed mutagenesis, we observed that residues in transmembrane (TM) segments 1, 4, and 6 contribute to GlcCer selection, with a conserved glutamine in the center of TM4 playing an essential role. Our molecular observations help refine models for substrate translocation by P4-ATPases, clarify the relationship between these flippases and human disease, and have fundamental implications for membrane organization and sphingolipid homeostasis.


Subject(s)
ATP-Binding Cassette Transporters/chemistry , Adenosine Triphosphatases/chemistry , Membrane Transport Proteins/chemistry , Models, Molecular , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae/enzymology , Schizosaccharomyces pombe Proteins/chemistry , Schizosaccharomyces/enzymology , ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/metabolism , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Biological Transport, Active , Glucosylceramides/chemistry , Glucosylceramides/metabolism , HeLa Cells , Humans , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , Mutagenesis, Site-Directed , Protein Domains , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Schizosaccharomyces pombe Proteins/metabolism
9.
Crit Rev Biochem Mol Biol ; 51(6): 513-527, 2016.
Article in English | MEDLINE | ID: mdl-27696908

ABSTRACT

Cellular membranes display a diversity of functions that are conferred by the unique composition and organization of their proteins and lipids. One important aspect of lipid organization is the asymmetric distribution of phospholipids (PLs) across the plasma membrane. The unequal distribution of key PLs between the cytofacial and exofacial leaflets of the bilayer creates physical surface tension that can be used to bend the membrane; and like Ca2+, a chemical gradient that can be used to transduce biochemical signals. PL flippases in the type IV P-type ATPase (P4-ATPase) family are the principle transporters used to set and repair this PL gradient and the asymmetric organization of these membranes are encoded by the substrate specificity of these enzymes. Thus, understanding the mechanisms of P4-ATPase substrate specificity will help reveal their role in membrane organization and cell biology. Further, decoding the structural determinants of substrate specificity provides investigators the opportunity to mutationally tune this specificity to explore the role of particular PL substrates in P4-ATPase cellular functions. This work reviews the role of P4-ATPases in membrane biology, presents our current understanding of P4-ATPase substrate specificity, and discusses how these fundamental aspects of P4-ATPase enzymology may be used to enhance our knowledge of cellular membrane biology.


Subject(s)
Adenosine Triphosphatases/metabolism , Cell Membrane/metabolism , Phospholipid Transfer Proteins/metabolism , Phospholipids/metabolism , Adenosine Triphosphatases/chemistry , Animals , Cell Membrane/chemistry , Humans , Models, Molecular , Protein Domains , Substrate Specificity
10.
Proc Natl Acad Sci U S A ; 113(31): E4460-6, 2016 08 02.
Article in English | MEDLINE | ID: mdl-27432949

ABSTRACT

Phospholipid flippases in the type IV P-type ATPase (P4-ATPases) family establish membrane asymmetry and play critical roles in vesicular transport, cell polarity, signal transduction, and neurologic development. All characterized P4-ATPases flip glycerophospholipids across the bilayer to the cytosolic leaflet of the membrane, but how these enzymes distinguish glycerophospholipids from sphingolipids is not known. We used a directed evolution approach to examine the molecular mechanisms through which P4-ATPases discriminate substrate backbone. A mutagenesis screen in the yeast Saccharomyces cerevisiae has identified several gain-of-function mutations in the P4-ATPase Dnf1 that facilitate the transport of a novel lipid substrate, sphingomyelin. We found that a highly conserved asparagine (N220) in the first transmembrane segment is a key enforcer of glycerophospholipid selection, and specific substitutions at this site allow transport of sphingomyelin.


Subject(s)
ATP-Binding Cassette Transporters/genetics , Adenosine Triphosphatases/genetics , Gain of Function Mutation , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/genetics , Sphingomyelins/metabolism , ATP-Binding Cassette Transporters/metabolism , Adenosine Triphosphatases/metabolism , Amino Acid Sequence , Asparagine/genetics , Asparagine/metabolism , Biological Transport/genetics , Cell Membrane/metabolism , Directed Molecular Evolution , Mutagenesis , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae Proteins/metabolism , Sequence Homology, Amino Acid , Substrate Specificity
11.
PLoS Genet ; 12(3): e1005941, 2016 Mar.
Article in English | MEDLINE | ID: mdl-27031109

ABSTRACT

Triosephosphate isomerase (TPI) deficiency is a poorly understood disease characterized by hemolytic anemia, cardiomyopathy, neurologic dysfunction, and early death. TPI deficiency is one of a group of diseases known as glycolytic enzymopathies, but is unique for its severe patient neuropathology and early mortality. The disease is caused by missense mutations and dysfunction in the glycolytic enzyme, TPI. Previous studies have detailed structural and catalytic changes elicited by disease-associated TPI substitutions, and samples of patient erythrocytes have yielded insight into patient hemolytic anemia; however, the neuropathophysiology of this disease remains a mystery. This study combines structural, biochemical, and genetic approaches to demonstrate that perturbations of the TPI dimer interface are sufficient to elicit TPI deficiency neuropathogenesis. The present study demonstrates that neurologic dysfunction resulting from TPI deficiency is characterized by synaptic vesicle dysfunction, and can be attenuated with catalytically inactive TPI. Collectively, our findings are the first to identify, to our knowledge, a functional synaptic defect in TPI deficiency derived from molecular changes in the TPI dimer interface.


Subject(s)
Anemia, Hemolytic, Congenital Nonspherocytic/genetics , Carbohydrate Metabolism, Inborn Errors/genetics , Drosophila melanogaster/genetics , Nervous System Diseases/genetics , Synaptic Vesicles/genetics , Triose-Phosphate Isomerase/deficiency , Triose-Phosphate Isomerase/genetics , Anemia, Hemolytic, Congenital Nonspherocytic/pathology , Animals , Behavior, Animal , Carbohydrate Metabolism, Inborn Errors/pathology , Crystallography, X-Ray , Dimerization , Humans , Mutation, Missense , Nervous System Diseases/pathology , Protein Conformation , Synaptic Vesicles/pathology , Triose-Phosphate Isomerase/chemistry , Triose-Phosphate Isomerase/metabolism
12.
Biochim Biophys Acta ; 1852(1): 61-9, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25463631

ABSTRACT

Triosephosphate isomerase (TPI) is a glycolytic enzyme which homodimerizes for full catalytic activity. Mutations of the TPI gene elicit a disease known as TPI Deficiency, a glycolytic enzymopathy noted for its unique severity of neurological symptoms. Evidence suggests that TPI Deficiency pathogenesis may be due to conformational changes of the protein, likely affecting dimerization and protein stability. In this report, we genetically and physically characterize a human disease-associated TPI mutation caused by an I170V substitution. Human TPI(I170V) elicits behavioral abnormalities in Drosophila. An examination of hTPI(I170V) enzyme kinetics revealed this substitution reduced catalytic turnover, while assessments of thermal stability demonstrated an increase in enzyme stability. The crystal structure of the homodimeric I170V mutant reveals changes in the geometry of critical residues within the catalytic pocket. Collectively these data reveal new observations of the structural and kinetic determinants of TPI Deficiency pathology, providing new insights into disease pathogenesis.


Subject(s)
Anemia, Hemolytic, Congenital Nonspherocytic/pathology , Carbohydrate Metabolism, Inborn Errors/pathology , Catalytic Domain , Triose-Phosphate Isomerase/deficiency , Triose-Phosphate Isomerase/metabolism , Anemia, Hemolytic, Congenital Nonspherocytic/enzymology , Animals , Behavior, Animal , Carbohydrate Metabolism, Inborn Errors/enzymology , Disease Models, Animal , Drosophila , Enzyme Stability , Humans , Mutation , Triose-Phosphate Isomerase/chemistry , Triose-Phosphate Isomerase/genetics
13.
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
14.
J Cell Sci ; 126(Pt 14): 3151-8, 2013 Jul 15.
Article in English | MEDLINE | ID: mdl-23641070

ABSTRACT

Triosephosphate isomerase (TPI) is a glycolytic enzyme that converts dihydroxyacetone phosphate (DHAP) into glyceraldehyde 3-phosphate (GAP). Glycolytic enzyme dysfunction leads to metabolic diseases collectively known as glycolytic enzymopathies. Of these enzymopathies, TPI deficiency is unique in the severity of neurological symptoms. The Drosophila sugarkill mutant closely models TPI deficiency and encodes a protein prematurely degraded by the proteasome. This led us to question whether enzyme catalytic activity was crucial to the pathogenesis of TPI sugarkill neurological phenotypes. To study TPI deficiency in vivo we developed a genomic engineering system for the TPI locus that enables the efficient generation of novel TPI genetic variants. Using this system we demonstrate that TPI sugarkill can be genetically complemented by TPI encoding a catalytically inactive enzyme. Furthermore, our results demonstrate a non-metabolic function for TPI, the loss of which contributes significantly to the neurological dysfunction in this animal model.


Subject(s)
Anemia, Hemolytic, Congenital Nonspherocytic/enzymology , Carbohydrate Metabolism, Inborn Errors/enzymology , Drosophila melanogaster/physiology , Longevity , Paralysis/enzymology , Triose-Phosphate Isomerase/deficiency , Triose-Phosphate Isomerase/metabolism , Anemia, Hemolytic, Congenital Nonspherocytic/genetics , Animals , Carbohydrate Metabolism, Inborn Errors/genetics , Catalysis , Dihydroxyacetone Phosphate/metabolism , Disease Models, Animal , Drosophila melanogaster/enzymology , Female , Gene Knockout Techniques , Genetic Complementation Test , Genetic Engineering , Glyceraldehyde 3-Phosphate/metabolism , Glycolysis/genetics , Hot Temperature/adverse effects , Male , Mutation/genetics , Paralysis/genetics , Stress, Physiological/genetics , Transgenes/genetics , Triose-Phosphate Isomerase/genetics
15.
J Mol Biol ; 415(5): 900-17, 2012 Feb 03.
Article in English | MEDLINE | ID: mdl-22178478

ABSTRACT

Although oligomeric intermediates are transiently formed in almost all known amyloid assembly reactions, their mechanistic roles are poorly understood. Recently, we demonstrated a critical role for the 17-amino-acid N-terminus (htt(NT) segment) of huntingtin (htt) in the oligomer-mediated amyloid assembly of htt N-terminal fragments. In this mechanism, the htt(NT) segment forms the α-helix-rich core of the oligomers, leaving much of the polyglutamine (polyQ) segment disordered and solvent-exposed. Nucleation of amyloid structure occurs within this local high concentration of disordered polyQ. Here we demonstrate the kinetic importance of htt(NT) self-assembly by describing inhibitory htt(NT)-containing peptides that appear to work by targeting nucleation within the oligomer fraction. These molecules inhibit amyloid nucleation by forming mixed oligomers with the htt(NT) domains of polyQ-containing htt N-terminal fragments. In one class of inhibitors, nucleation is passively suppressed due to the reduced local concentration of polyQ within the mixed oligomer. In the other class, nucleation is actively suppressed by a proline-rich polyQ segment covalently attached to htt(NT). Studies with D-amino acid and scrambled sequence versions of htt(NT) suggest that inhibition activity is strongly linked to the propensity of inhibitory peptides to make amphipathic α-helices. Htt(NT) derivatives with C-terminal cell-penetrating peptide segments also exhibit excellent inhibitory activity. The htt(NT)-based peptides described here, especially those with protease-resistant d-amino acids and/or with cell-penetrating sequences, may prove useful as lead therapeutics for inhibiting the nucleation of amyloid formation in Huntington's disease.


Subject(s)
Amyloid/chemical synthesis , Nerve Tissue Proteins/chemical synthesis , Nuclear Proteins/chemical synthesis , Polymers/chemical synthesis , Amino Acid Sequence , Amino Acids/chemistry , Amyloid/antagonists & inhibitors , Amyloid/genetics , Cell Line, Tumor , Cell-Penetrating Peptides/chemistry , Humans , Huntingtin Protein , Nerve Tissue Proteins/antagonists & inhibitors , Nerve Tissue Proteins/genetics , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/genetics , Peptides/chemistry , Protein Structure, Secondary
16.
Behav Brain Res ; 214(2): 193-200, 2010 Dec 25.
Article in English | MEDLINE | ID: mdl-20493905

ABSTRACT

The purpose of this study was to evaluate the therapeutic effects of the transplantation of bone-marrow mesenchymal stem cells (MSCs), genetically engineered to over-express brain-derived neurotrophic factor (BDNF) or nerve growth factor (NGF) on motor deficits and neurodegeneration in YAC 128 transgenic mice. MSCs, harvested from mouse femurs, were genetically engineered to over-express BDNF and/or NGF and these cells, or the vehicle solution, were injected into the striata of four-month old YAC 128 transgenic and wild-type mice. Assessments of motor ability on the rotarod and the severity of clasping were made one day prior to transplantation and once monthly, thereafter, to determine the effects of the transplanted cells on motor function. The mice were sacrificed at 13-months of age for immunohistological examination. All YAC 128 mice receiving transplants had reduced clasping, relative to vehicle-treated YAC 128 mice, while YAC 128 mice that were transplanted with MSCs which were genetically engineered to over-express BDNF, had the longest latencies on the rotarod and the least amount of neuronal loss within the striatum of the YAC 128 mice. These results indicate that intrastriatal transplantation of MSCs that over-express BDNF may create an environment within the striatum that slows neurodegenerative processes and provides behavioral sparing in the YAC 128 mouse model of HD. Further research on the long-term safety and efficacy of this approach is needed before its potential clinical utility can be comprehensively assessed.


Subject(s)
Chromosomes, Artificial, Yeast/genetics , Genetic Engineering/methods , Huntington Disease/therapy , Mesenchymal Stem Cell Transplantation/methods , Mesenchymal Stem Cells/metabolism , Motor Skills/physiology , Animals , Brain-Derived Neurotrophic Factor/metabolism , Corpus Striatum/pathology , Corpus Striatum/surgery , Disease Models, Animal , Huntington Disease/genetics , Huntington Disease/pathology , Mice , Mice, Transgenic , Nerve Growth Factor/metabolism
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