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1.
Cancer Res ; 2024 Jun 11.
Article in English | MEDLINE | ID: mdl-38861359

ABSTRACT

The NCI60 human tumor cell line screen has been in operation as a service to the cancer research community for over 30 years. The screen operated with 96-well plates, a 2-day exposure period to test agents, and, following cell fixation, a visible absorbance endpoint by the protein-staining dye sulforhodamine B. Here, we describe the next phase of this important cancer research tool, the HTS384 NCI60 screen. While the cell lines remain the same, the updated screen is performed with 384-well plates, a 3-day exposure period to test agents, and a luminescent endpoint to measure cell viability based upon cellular ATP content. In this study, a library of 1003 FDA-approved and investigational small molecule anticancer agents was screened by the two NCI60 assays. The datasets were compared with a focus on targeted agents with at least six representatives in the library. For many agents, including inhibitors of EGFR, BRAF, MEK, ERK, and PI3K, the patterns of GI50 values were very similar between the screens with strong correlations between those patterns within the dataset from each screen. However, for some groups of targeted agents, including mTOR, BET bromodomain, and NAMPRTase inhibitors, there were limited or no correlations between the two datasets, although the patterns of GI50 values and correlations between those patterns within each dataset were apparent. Beginning in January 2024, the HTS384 NCI60 screen became the free screening service of the National Cancer Institute to facilitate drug discovery by the cancer research community.

2.
Nat Biomed Eng ; 5(7): 657-665, 2021 07.
Article in English | MEDLINE | ID: mdl-34211145

ABSTRACT

Frequent and widespread testing of members of the population who are asymptomatic for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for the mitigation of the transmission of the virus. Despite the recent increases in testing capacity, tests based on quantitative polymerase chain reaction (qPCR) assays cannot be easily deployed at the scale required for population-wide screening. Here, we show that next-generation sequencing of pooled samples tagged with sample-specific molecular barcodes enables the testing of thousands of nasal or saliva samples for SARS-CoV-2 RNA in a single run without the need for RNA extraction. The assay, which we named SwabSeq, incorporates a synthetic RNA standard that facilitates end-point quantification and the calling of true negatives, and that reduces the requirements for automation, purification and sample-to-sample normalization. We used SwabSeq to perform 80,000 tests, with an analytical sensitivity and specificity comparable to or better than traditional qPCR tests, in less than two months with turnaround times of less than 24 h. SwabSeq could be rapidly adapted for the detection of other pathogens.


Subject(s)
RNA, Viral/genetics , SARS-CoV-2/pathogenicity , Saliva/virology , High-Throughput Nucleotide Sequencing , Humans , SARS-CoV-2/genetics , Sensitivity and Specificity
3.
medRxiv ; 2021 Mar 09.
Article in English | MEDLINE | ID: mdl-32909008

ABSTRACT

The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is due to the high rates of transmission by individuals who are asymptomatic at the time of transmission1,2. Frequent, widespread testing of the asymptomatic population for SARS-CoV-2 is essential to suppress viral transmission. Despite increases in testing capacity, multiple challenges remain in deploying traditional reverse transcription and quantitative PCR (RT-qPCR) tests at the scale required for population screening of asymptomatic individuals. We have developed SwabSeq, a high-throughput testing platform for SARS-CoV-2 that uses next-generation sequencing as a readout. SwabSeq employs sample-specific molecular barcodes to enable thousands of samples to be combined and simultaneously analyzed for the presence or absence of SARS-CoV-2 in a single run. Importantly, SwabSeq incorporates an in vitro RNA standard that mimics the viral amplicon, but can be distinguished by sequencing. This standard allows for end-point rather than quantitative PCR, improves quantitation, reduces requirements for automation and sample-to-sample normalization, enables purification-free detection, and gives better ability to call true negatives. After setting up SwabSeq in a high-complexity CLIA laboratory, we performed more than 80,000 tests for COVID-19 in less than two months, confirming in a real world setting that SwabSeq inexpensively delivers highly sensitive and specific results at scale, with a turn-around of less than 24 hours. Our clinical laboratory uses SwabSeq to test both nasal and saliva samples without RNA extraction, while maintaining analytical sensitivity comparable to or better than traditional RT-qPCR tests. Moving forward, SwabSeq can rapidly scale up testing to mitigate devastating spread of novel pathogens.

4.
Elife ; 92020 10 21.
Article in English | MEDLINE | ID: mdl-33084570

ABSTRACT

The >800 human G protein-coupled receptors (GPCRs) are responsible for transducing diverse chemical stimuli to alter cell state- and are the largest class of drug targets. Their myriad structural conformations and various modes of signaling make it challenging to understand their structure and function. Here, we developed a platform to characterize large libraries of GPCR variants in human cell lines with a barcoded transcriptional reporter of G protein signal transduction. We tested 7800 of 7828 possible single amino acid substitutions to the beta-2 adrenergic receptor (ß2AR) at four concentrations of the agonist isoproterenol. We identified residues specifically important for ß2AR signaling, mutations in the human population that are potentially loss of function, and residues that modulate basal activity. Using unsupervised learning, we identify residues critical for signaling, including all major structural motifs and molecular interfaces. We also find a previously uncharacterized structural latch spanning the first two extracellular loops that is highly conserved across Class A GPCRs and is conformationally rigid in both the inactive and active states of the receptor. More broadly, by linking deep mutational scanning with engineered transcriptional reporters, we establish a generalizable method for exploring pharmacogenomics, structure and function across broad classes of drug receptors.


Subject(s)
DNA Mutational Analysis/methods , Receptors, G-Protein-Coupled/chemistry , Cloning, Molecular , DNA Barcoding, Taxonomic , Gene Editing , HEK293 Cells , Humans , Machine Learning , Models, Molecular , Protein Conformation , Receptors, G-Protein-Coupled/metabolism
5.
Cell Syst ; 11(1): 75-85.e7, 2020 07 22.
Article in English | MEDLINE | ID: mdl-32603702

ABSTRACT

In eukaryotes, transcription factors (TFs) orchestrate gene expression by binding to TF-binding sites (TFBSs) and localizing transcriptional co-regulators and RNA polymerase II to cis-regulatory elements. However, we lack a basic understanding of the relationship between TFBS composition and their quantitative transcriptional responses. Here, we measured expression driven by 17,406 synthetic cis-regulatory elements with varied compositions of a model TFBS, the c-AMP response element (CRE) by using massively parallel reporter assays (MPRAs). We find CRE number, affinity, and promoter proximity largely determines expression. In addition, we observe expression modulation based on the spacing between CREs and CRE distance to the promoter, where expression follows a helical periodicity. Finally, we compare library expression between an episomal MPRA and a genomically integrated MPRA, where a single cis-regulatory element is assayed per cell at a defined locus. These assays largely recapitulate each other, although weaker, non-canonical CREs exhibit greater activity in a genomic context.


Subject(s)
Adenosine Monophosphate/metabolism , Genomics/methods , Plasmids/metabolism , Response Elements/genetics , Humans
6.
Cell Syst ; 8(3): 254-260.e6, 2019 03 27.
Article in English | MEDLINE | ID: mdl-30904378

ABSTRACT

G protein-coupled receptors (GPCRs) are central to how mammalian cells sense and respond to chemicals. Mammalian olfactory receptors (ORs), the largest family of GPCRs, mediate the sense of smell through activation by small molecules, though for most bonafide ligands, they have not been identified. Here, we introduce a platform to screen large chemical panels against multiplexed GPCR libraries using next-generation sequencing of barcoded genetic reporters in stably engineered human cell lines. We mapped 39 mammalian ORs against 181 odorants and identified 79 interactions that have not been reported to our knowledge, including ligands for 15 previously orphaned receptors. This multiplexed receptor assay allows the cost-effective mapping of large chemical libraries to receptor repertoires at scale.


Subject(s)
Odorants , Receptors, Odorant/metabolism , Sequence Analysis, RNA/methods , Signal Transduction , Smell , Animals , Cell Line , Gene Expression Profiling , Humans , Ligands , Mammals/metabolism , Mammals/physiology
7.
FEBS Lett ; 593(5): 518-526, 2019 03.
Article in English | MEDLINE | ID: mdl-30756384

ABSTRACT

Amyloid formation occurs via numerous complex mechanisms, often involving intermediates. This study examines the mechanism of amyloidogenesis in two N-terminal fragments of serum amyloid A (SAA), which are known to exhibit dramatically different amyloid structures. Fibrillization kinetics by these peptides are found to exhibit two unusual features: slower rates at higher peptide concentration, and complete insensitivity to addition of pre-formed seed. Additionally, we find that these peptides form micelle-like oligomers in solution. Our results imply an unusual dual role of micellar oligomers in amyloidogenesis, in which these particles act both as an off-pathway reservoir of peptide, and an inhibitory aggregate that slows amyloid growth. We anticipate that this mechanism of fibril formation may exist in other hydrophobic amyloid-forming peptides and proteins.


Subject(s)
Amyloid beta-Peptides/metabolism , Biopolymers/metabolism , Micelles , Protein Multimerization , Serum Amyloid A Protein/metabolism , Amyloid beta-Peptides/chemistry , Hydrophobic and Hydrophilic Interactions , Kinetics , Protein Binding , Serum Amyloid A Protein/chemistry
8.
Mol Cell ; 73(1): 183-194.e8, 2019 01 03.
Article in English | MEDLINE | ID: mdl-30503770

ABSTRACT

Mutations that lead to splicing defects can have severe consequences on gene function and cause disease. Here, we explore how human genetic variation affects exon recognition by developing a multiplexed functional assay of splicing using Sort-seq (MFASS). We assayed 27,733 variants in the Exome Aggregation Consortium (ExAC) within or adjacent to 2,198 human exons in the MFASS minigene reporter and found that 3.8% (1,050) of variants, most of which are extremely rare, led to large-effect splice-disrupting variants (SDVs). Importantly, we find that 83% of SDVs are located outside of canonical splice sites, are distributed evenly across distinct exonic and intronic regions, and are difficult to predict a priori. Our results indicate extant, rare genetic variants can have large functional effects on splicing at appreciable rates, even outside the context of disease, and MFASS enables their empirical assessment at scale.


Subject(s)
Exons , Gene Expression Profiling/methods , High-Throughput Nucleotide Sequencing/methods , Mutation , RNA Splicing , Sequence Analysis, DNA/methods , Cell Separation , Computational Biology , Flow Cytometry , HEK293 Cells , HeLa Cells , Hep G2 Cells , Humans , Introns , K562 Cells , Oligonucleotide Array Sequence Analysis , Reproducibility of Results
9.
J Phys Chem B ; 120(39): 10258-10268, 2016 10 06.
Article in English | MEDLINE | ID: mdl-27632709

ABSTRACT

Amyloid polymorphism presents a challenge to physical theories of amyloid formation and stability. The amyloidogenic protein serum amyloid A (SAA) exhibits complex and unexplained structural polymorphism in its N-terminal fragments: the N-terminal 11-residue peptide (SAA1-11) forms left-handed helical fibrils, while extension by one residue (SAA1-12) produces a rare right-handed amyloid. In this study, we use a combination of vibrational spectroscopy and ultramicroscopy to examine fibrils of these peptides and their terminally acetylated and amidated variants, in an effort to uncover the physical basis for this effect. Raman spectroscopy and atomic force microscopy provide evidence that SAA1-12 forms a ß-helical fibril architecture, while SAA1-11 forms more typical stacked ß-sheets. Importantly, N-terminal acetylation blocks fibril formation by SAA1-12 with no effect on SAA1-11, while C-terminal amidation has nearly the opposite effect. Together, these data suggest distinct electrostatic interactions at the N- and C-termini stabilize the two fibril structures; we propose model fibril structures in which C-terminal extension changes the favored intermolecular interaction between peptide monomers from an Arg1-C-terminus charge pair to an N-terminus-C-terminus charge pair. This model suggests a general mechanism for charge-mediated amyloid polymorphism and may inform strategies for design of peptide-based nanomaterials stabilized by engineered intermolecular contacts.


Subject(s)
Peptide Fragments/chemistry , Serum Amyloid A Protein/chemistry , Static Electricity , Peptide Fragments/chemical synthesis , Protein Conformation , Serum Amyloid A Protein/chemical synthesis
10.
Front Psychol ; 6: 919, 2015.
Article in English | MEDLINE | ID: mdl-26191026

ABSTRACT

Immersive software tools are virtual environments designed to give their users an augmented view of real-world data and ways of manipulating that data. As virtual environments, every action users make while interacting with these tools can be carefully logged, as can the state of the software and the information it presents to the user, giving these actions context. This data provides a high-resolution lens through which dynamic cognitive and behavioral processes can be viewed. In this report, we describe new methods for the analysis and interpretation of such data, utilizing a novel implementation of the Beta Process Hidden Markov Model (BP-HMM) for analysis of software activity logs. We further report the results of a preliminary study designed to establish the validity of our modeling approach. A group of 20 participants were asked to play a simple computer game, instrumented to log every interaction with the interface. Participants had no previous experience with the game's functionality or rules, so the activity logs collected during their naïve interactions capture patterns of exploratory behavior and skill acquisition as they attempted to learn the rules of the game. Pre- and post-task questionnaires probed for self-reported styles of problem solving, as well as task engagement, difficulty, and workload. We jointly modeled the activity log sequences collected from all participants using the BP-HMM approach, identifying a global library of activity patterns representative of the collective behavior of all the participants. Analyses show systematic relationships between both pre- and post-task questionnaires, self-reported approaches to analytic problem solving, and metrics extracted from the BP-HMM decomposition. Overall, we find that this novel approach to decomposing unstructured behavioral data within software environments provides a sensible means for understanding how users learn to integrate software functionality for strategic task pursuit.

11.
J Am Chem Soc ; 136(52): 18044-51, 2014 Dec 31.
Article in English | MEDLINE | ID: mdl-25474758

ABSTRACT

Amyloid fibers, once exclusively associated with disease, are acquiring utility as a class of biological nanomaterials. Here we introduce a method that utilizes the atomic structures of amyloid peptides, to design materials with versatile applications. As a model application, we designed amyloid fibers capable of capturing carbon dioxide from flue gas, to address the global problem of excess anthropogenic carbon dioxide. By measuring dynamic separation of carbon dioxide from nitrogen, we show that fibers with designed amino acid sequences double the carbon dioxide binding capacity of the previously reported fiber formed by VQIVYK from Tau protein. In a second application, we designed fibers that facilitate retroviral gene transfer. By measuring lentiviral transduction, we show that designed fibers exceed the efficiency of polybrene, a commonly used enhancer of transduction. The same procedures can be adapted to the design of countless other amyloid materials with a variety of properties and uses.


Subject(s)
Amyloid/chemistry , Drug Design , Amino Acid Sequence , Carbon Dioxide/chemistry , Carbon Dioxide/isolation & purification , HEK293 Cells , HIV-1/genetics , Humans , Models, Molecular , Peptide Fragments/chemistry , Peptide Fragments/pharmacology , Protein Structure, Secondary , Transduction, Genetic
12.
J Am Chem Soc ; 136(29): 10325-39, 2014 Jul 23.
Article in English | MEDLINE | ID: mdl-24991732

ABSTRACT

The use of hybrid hemoglobin (Hb), with mesoheme substituted for protoheme, allows separate monitoring of the α or ß hemes along the allosteric pathway. Using resonance Raman (rR) spectroscopy in silica gel, which greatly slows protein motions, we have observed that the Fe-histidine stretching frequency, νFeHis, which is a monitor of heme reactivity, evolves between frequencies characteristic of the R and T states, for both α or ß chains, prior to the quaternary R-T and T-R shifts. Computation of νFeHis, using QM/MM and the conformational search program PELE, produced remarkable agreement with experiment. Analysis of the PELE structures showed that the νFeHis shifts resulted from heme distortion and, in the α chain, Fe-His bond tilting. These results support the tertiary two-state model of ligand binding (Henry et al., Biophys. Chem. 2002, 98, 149). Experimentally, the νFeHis evolution is faster for ß than for α chains, and pump-probe rR spectroscopy in solution reveals an inflection in the νFeHis time course at 3 µs for ß but not for α hemes, an interval previously shown to be the first step in the R-T transition. In the α chain νFeHis dropped sharply at 20 µs, the final step in the R-T transition. The time courses are fully consistent with recent computational mapping of the R-T transition via conjugate peak refinement by Karplus and co-workers (Fischer et al., Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 5608). The effector molecule IHP was found to lower νFeHis selectively for α chains within the R state, and a binding site in the α1α2 cleft is suggested.


Subject(s)
Computational Biology/methods , Heme/chemistry , Hemoglobin A/chemistry , Spectrum Analysis, Raman/methods , alpha-Globins/chemistry , beta-Globins/chemistry , Allosteric Regulation , Mesoporphyrins/chemistry , Models, Molecular , Protein Structure, Quaternary
13.
Protein Sci ; 23(8): 1094-101, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24895328

ABSTRACT

Calmodulin (CaM) is a ubiquitous moderator protein for calcium signaling in all eukaryotic cells. This small calcium-binding protein exhibits a broad range of structural transitions, including domain opening and folding-unfolding, that allow it to recognize a wide variety of binding partners in vivo. While the static structures of CaM associated with its various binding activities are fairly well-known, it has been challenging to examine the dynamics of transition between these structures in real-time, due to a lack of suitable spectroscopic probes of CaM structure. In this article, we examine the potential of ultraviolet resonance Raman (UVRR) spectroscopy for clarifying the nature of structural transitions in CaM. We find that the UVRR spectral change (with 229 nm excitation) due to thermal unfolding of CaM is qualitatively different from that associated with opening of the C-terminal domain in response to Ca(2+) binding. This spectral difference is entirely due to differences in tertiary contacts at the interdomain tyrosine residue Tyr138, toward which other spectroscopic methods are not sensitive. We conclude that UVRR is ideally suited to identifying the different types of structural transitions in CaM and other proteins with conformation-sensitive tyrosine residues, opening a path to time-resolved studies of CaM dynamics using Raman spectroscopy.


Subject(s)
Calmodulin/chemistry , Protein Unfolding , Calmodulin/metabolism , Models, Molecular , Protein Conformation , Spectrum Analysis, Raman , Temperature
14.
J Clin Exp Neuropsychol ; 34(9): 919-24, 2012.
Article in English | MEDLINE | ID: mdl-22794076

ABSTRACT

Whereas the ventral visual processing stream mediates facial and object recognition, the dorsal stream mediates recognition of spatial relationships. In addition, ventral lesions have been reported to induce visual inattention to the upper visual field and dorsal lesions inattention to the lower field. The purpose of this study is to test the hypothesis that activation of the ventral stream will induce an upward attentional bias and activation of the dorsal stream, a downward bias, as assessed by vertical line bisection tests. Twelve healthy right-handed individuals performed vertical line bisections. During these trials, either pictures of famous faces or dots in different spatial locations were presented above and below the line. The participants were asked to recognize and remember the faces or locations of dots while they performed the bisections. In control trials, they were no faces or dots. An upward bias was observed in all conditions. This upward bias was significantly increased in the face recognition and recall condition, but not altered in the dot location condition. Although the face task appeared to activate the ventral stream and increase the upward vertical bias, the failure of the dot localization task to alter the bias may be related task selection. Dorsolateral lesions cause optic ataxia, a disorder of the egocentric "where" system, and the dot location task in this study was allocentric. Thus, further research will be needed to learn whether an egocentric spatial localization task, with a memory component, will alter the vertical attentional bias.


Subject(s)
Attention/physiology , Pattern Recognition, Visual/physiology , Recognition, Psychology/physiology , Space Perception/physiology , Visual Pathways/physiology , Adolescent , Analysis of Variance , Face , Female , Humans , Male , Reference Values , Young Adult
15.
J Am Chem Soc ; 134(7): 3461-71, 2012 Feb 22.
Article in English | MEDLINE | ID: mdl-22263778

ABSTRACT

Encapsulation of hemoglobin (Hb) in silica gel preserves structure and function but greatly slows protein motion, thereby providing access to intermediates along the allosteric pathway that are inaccessible in solution. Resonance Raman (RR) spectroscopy with visible and ultraviolet laser excitation provides probes of heme reactivity and of key tertiary and quaternary contacts. These probes were monitored in gels after deoxygenation of oxyHb and after CO binding to deoxyHb, which initiate conformational change in the R-T and T-R directions, respectively. The spectra establish that quaternary structure change in the gel takes a week or more but that the evolution of heme reactivity, as monitored by the Fe-histidine stretching vibration, ν(FeHis), is completed within two days, and is therefore uncoupled from the quaternary structure. Within each quaternary structure, the evolving ν(FeHis) frequencies span the full range of values between those previously associated with the high- and low-affinity end states, R and T. This result supports the tertiary two-state (TTS) model, in which the Hb subunits can adopt high- and low-affinity tertiary structures, r and t, within each quaternary state. The spectra also reveal different tertiary pathways, involving the breaking and reformation of E and F interhelical contacts in the R-T direction but not the T-R direction. In the latter, tertiary motions are restricted by the T quaternary contacts.


Subject(s)
Heme/metabolism , Hemoglobin A/metabolism , Immobilized Proteins/metabolism , Spectrum Analysis, Raman/methods , Carbon Monoxide/metabolism , Heme/chemistry , Hemoglobin A/chemistry , Humans , Immobilized Proteins/chemistry , Models, Molecular , Protein Structure, Tertiary
16.
J Biol Chem ; 286(49): 42777-42784, 2011 Dec 09.
Article in English | MEDLINE | ID: mdl-22002245

ABSTRACT

The C-terminally-truncated human prion protein variant Y145Stop (or PrP23-144), associated with a familial prion disease, provides a valuable model for studying the fundamental properties of protein amyloids. In previous solid-state NMR experiments, we established that the ß-sheet core of the PrP23-144 amyloid is composed of two ß-strand regions encompassing residues ∼113-125 and ∼130-140. The former segment contains a highly conserved hydrophobic palindrome sequence, (113)AGAAAAGA(120), which has been considered essential to PrP conformational conversion. Here, we examine the role of this segment in fibrillization of PrP23-144 using a deletion variant, Δ113-120 PrP23-144, in which the palindrome sequence is missing. Surprisingly, we find that deletion of the palindrome sequence affects neither the amyloidogenicity nor the polymerization kinetics of PrP23-144, although it does alter amyloid conformation and morphology. Using two-dimensional and three-dimensional solid-state NMR methods, we find that Δ113-120 PrP23-144 fibrils contain an altered ß-core extended N-terminally to residue ∼106, encompassing residues not present in the core of wild-type PrP23-144 fibrils. The C-terminal ß-strand of the core, however, is similar in both fibril types. Collectively, these data indicate that amyloid cores of PrP23-144 variants contain "essential" (i.e. nucleation-determining) and "nonessential" regions, with the latter being "movable" in amino acid sequence space. These findings reveal an intriguing new mechanism for structural polymorphism in amyloids and suggest a potential means for modulating the physicochemical properties of amyloid fibrils without compromising their polymerization characteristics.


Subject(s)
Amyloid/chemistry , Amyloid/genetics , Polymorphism, Genetic , Prions/chemistry , Escherichia coli/metabolism , Gene Deletion , Humans , Kinetics , Magnetic Resonance Spectroscopy/methods , Microscopy, Atomic Force/methods , Prion Diseases/metabolism , Protein Denaturation , Protein Folding , Protein Structure, Secondary , Proteins/chemistry , Spectroscopy, Fourier Transform Infrared/methods
17.
Biophys J ; 95(11): 5268-80, 2008 Dec.
Article in English | MEDLINE | ID: mdl-18723592

ABSTRACT

Oxidation of methionine residues in calmodulin (CaM) lowers the affinity for calcium and results in an inability to activate target proteins fully. To evaluate the structural consequences of CaM oxidation, we used infrared difference spectroscopy to identify oxidation-dependent effects on protein conformation and calcium liganding. Oxidation-induced changes include an increase in hydration of alpha-helices, as indicated in the downshift of the amide I' band of both apo-CaM and Ca(2+)-CaM, and a modification of calcium liganding by carboxylate side chains, reflected in antisymmetric carboxylate band shifts. Changes in carboxylate ligands are consistent with the model we propose: an Asp at position 1 of the EF-loop experiences diminished hydrogen bonding with the polypeptide backbone, an Asp at position 3 forms a bidentate coordination of calcium, and an Asp at position 5 forms a pseudobridging coordination with a calcium-bound water molecule. The bidentate coordination of calcium by conserved glutamates is unaffected by oxidation. The observed changes in calcium ligation are discussed in terms of the placement of methionine side chains relative to the calcium-binding sites, suggesting that varying sensitivities of binding sites to oxidation may underlie the loss of CaM function upon oxidation.


Subject(s)
Calcium/metabolism , Calmodulin/chemistry , Calmodulin/metabolism , Methionine/metabolism , Absorption , Amino Acid Sequence , Animals , Binding Sites , Carboxylic Acids , Ligands , Molecular Sequence Data , Oxidation-Reduction , Protein Stability , Protein Structure, Secondary , Spectroscopy, Fourier Transform Infrared
18.
Acc Chem Res ; 39(9): 654-62, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16981682

ABSTRACT

Self-perpetuating conformational conversion of the cellular prion protein PrP(C) into the beta-sheet-rich "scrapie" conformer (PrP(Sc)) is believed to be the central molecular event in pathogenesis of a group of diseases known as transmissible spongiform encephalopathies. Recent advances provide growing support for the notion that a misfolded protein alone might act as an infectious agent. Furthermore, findings regarding the mechanism of prion protein structural rearrangement, the role of folding intermediates in conformational conversion, and "conformational adaptability" in the propagation of prion amyloids in vitro yield molecular-level insight into such phenomena as inherited prion diseases, prion transmission barriers, and prion strains.


Subject(s)
Prions , Animals , Biophysical Phenomena , Biophysics , Models, Molecular , PrPC Proteins/chemistry , PrPC Proteins/metabolism , PrPSc Proteins/chemistry , PrPSc Proteins/metabolism , Protein Conformation
19.
J Biol Chem ; 281(12): 8190-6, 2006 Mar 24.
Article in English | MEDLINE | ID: mdl-16443601

ABSTRACT

A self-perpetuating conformational conversion of the prion protein (PrP) is believed to underlie pathology and transmission of prion diseases. Here we explore the effects of N-terminal pathogenic mutations (P102L, P105L, A117V) and the residue 129 polymorphism on amyloid fibril formation by the human PrP fragment 23-144, an in vitro conversion model that can reproduce certain characteristics of prion replication such as strains and species barriers. We find that these amino acid substitutions neither affect PrP23-144 amyloidogenicity nor introduce barriers to cross-seeding of soluble protein. However, the polymorphism strongly influences the conformation of the amyloid fibrils, as determined by infrared spectroscopy. Intriguingly, unlike conformational features governed by the critical amyloidogenic region of PrP23-144 (residues 138-139), the structural features distinguishing Met-129 and Val-129 PrP23-144 amyloid fibrils are not transmissible by cross-seeding. While based only on in vitro data, these findings provide fundamental insight into the mechanism of prion-based conformational transmission, indicating that only conformational features controlling seeding specificity (e.g. those in critical intermolecular contact sites of amyloid fibrils) are necessarily transmissible by cross-seeding; conformational traits in other parts of the PrP molecule may not be "heritable" from the amyloid template.


Subject(s)
Mutation , Prions/chemistry , Prions/genetics , Amyloid/chemistry , Benzothiazoles , Escherichia coli/metabolism , Genetic Predisposition to Disease/genetics , Humans , In Vitro Techniques , Kinetics , Methionine/chemistry , Microscopy, Atomic Force , Molecular Conformation , Plasmids/metabolism , Polymorphism, Genetic , Prion Diseases/genetics , Protein Binding , Protein Conformation , Protein Structure, Tertiary , Spectroscopy, Fourier Transform Infrared , Thiazoles/chemistry , Time Factors , Valine/chemistry
20.
Cell ; 121(1): 63-72, 2005 Apr 08.
Article in English | MEDLINE | ID: mdl-15820679

ABSTRACT

Spongiform encephalopathies are believed to be transmitted by self-perpetuating conformational conversion of the prion protein. It was shown recently that fundamental aspects of mammalian prion propagation can be reproduced in vitro in a seeded fibrillization of the recombinant prion protein variant Y145Stop (PrP23-144). Here we demonstrate that PrP23-144 amyloids from different species adopt distinct secondary structures and morphologies, and that these structural differences are controlled by one or two residues in a critical region. These sequence-specific structural characteristics correlate strictly with the seeding specificity of amyloid fibrils. However, cross-seeding of PrP23-144 from one species with preformed fibrils from another species may overcome natural sequence-based structural preferences, resulting in a new amyloid strain that inherits the secondary structure and morphology of the template. These data provide direct biophysical evidence that protein conformations are transmitted in PrP amyloid strains, establishing a foundation for a structural basis of mammalian prion transmission barriers.


Subject(s)
Amyloid/chemistry , Mutation/genetics , Prions/chemistry , Recombinant Proteins/chemistry , Amyloid/genetics , Amyloid/ultrastructure , Animals , Humans , Microscopy, Atomic Force , Prions/genetics , Prions/ultrastructure , Protein Conformation , Recombinant Proteins/genetics , Recombinant Proteins/ultrastructure , Species Specificity , Spectroscopy, Fourier Transform Infrared
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