Publications by authors named "Lisa N Kinch"

96 Publications

Target classification in the 14th round of the critical assessment of protein structure prediction (CASP14).

Proteins 2021 Aug 4. Epub 2021 Aug 4.

Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

An evolutionary-based definition and classification of target evaluation units (EUs) is presented for the 14th round of the critical assessment of structure prediction (CASP14). CASP14 targets included 84 experimental models submitted by various structural groups (designated T1024-T1101). Targets were split into EUs based on the domain organization of available templates and performance of server groups. Several targets required splitting (19 out of 25 multidomain targets) due in part to observed conformation changes. All in all, 96 CASP14 EUs were defined and assigned to tertiary structure assessment categories (Topology-based FM or High Accuracy-based TBM-easy and TBM-hard) considering their evolutionary relationship to existing ECOD fold space: 24 family level, 50 distant homologs (H-group), 12 analogs (X-group), and 10 new folds. Principal component analysis and heatmap visualization of sequence and structure similarity to known templates as well as performance of servers highlighted trends in CASP14 target difficulty. The assigned evolutionary levels (i.e., H-groups) and assessment classes (i.e., FM) displayed overlapping clusters of EUs. Many viral targets diverged considerably from their template homologs and thus were more difficult for prediction than other homology-related targets. On the other hand, some targets did not have sequence-identifiable templates, but were predicted better than expected due to relatively simple arrangements of secondary structural elements. An apparent improvement in overall server performance in CASP14 further complicated traditional classification, which ultimately assigned EUs into high-accuracy modeling (27 TBM-easy and 31 TBM-hard), topology (23 FM), or both (15 FM/TBM).
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http://dx.doi.org/10.1002/prot.26202DOI Listing
August 2021

Accurate prediction of protein structures and interactions using a three-track neural network.

Science 2021 08 15;373(6557):871-876. Epub 2021 Jul 15.

Molecular Biophysics & Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.

DeepMind presented notably accurate predictions at the recent 14th Critical Assessment of Structure Prediction (CASP14) conference. We explored network architectures that incorporate related ideas and obtained the best performance with a three-track network in which information at the one-dimensional (1D) sequence level, the 2D distance map level, and the 3D coordinate level is successively transformed and integrated. The three-track network produces structure predictions with accuracies approaching those of DeepMind in CASP14, enables the rapid solution of challenging x-ray crystallography and cryo-electron microscopy structure modeling problems, and provides insights into the functions of proteins of currently unknown structure. The network also enables rapid generation of accurate protein-protein complex models from sequence information alone, short-circuiting traditional approaches that require modeling of individual subunits followed by docking. We make the method available to the scientific community to speed biological research.
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http://dx.doi.org/10.1126/science.abj8754DOI Listing
August 2021

Topology evaluation of models for difficult targets in the 14th round of the critical assessment of protein structure prediction.

Proteins 2021 Jul 9. Epub 2021 Jul 9.

Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

This report describes the tertiary structure prediction assessment of difficult modeling targets in the 14th round of the Critical Assessment of Structure Prediction (CASP14). We implemented an official ranking scheme that used the same scores as the previous CASP topology-based assessment, but combined these scores with one that emphasized physically realistic models. The top performing AlphaFold2 group outperformed the rest of the prediction community on all but two of the difficult targets considered in this assessment. They provided high quality models for most of the targets (86% over GDT_TS 70), including larger targets above 150 residues, and they correctly predicted the topology of almost all the rest. AlphaFold2 performance was followed by two manual Baker methods, a Feig method that refined Zhang-server models, two notable automated Zhang server methods (QUARK and Zhang-server), and a Zhang manual group. Despite the remarkable progress in protein structure prediction of difficult targets, both the prediction community and AlphaFold2, to a lesser extent, faced challenges with flexible regions and obligate oligomeric assemblies. The official ranking of top-performing methods was supported by performance generated PCA and heatmap clusters that gave insight into target difficulties and the most successful state-of-the-art structure prediction methodologies.
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http://dx.doi.org/10.1002/prot.26172DOI Listing
July 2021

Completeness and Consistency in Structural Domain Classifications.

ACS Omega 2021 Jun 8;6(24):15698-15707. Epub 2021 Jun 8.

Departments of Biophysics and Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390, United States.

Domain classifications are a useful resource for computational analysis of the protein structure, but elements of their composition are often opaque to potential users. We perform a comparative analysis of our classification ECOD against the SCOPe, SCOP2, and CATH domain classifications with respect to their constituent domain boundaries and hierarchal organization. The coverage of these domain classifications with respect to ECOD and to the PDB was assessed by structure and by sequence. We also conducted domain pair analysis to determine broad differences in hierarchy between domains shared by ECOD and other classifications. Finally, we present domains from the major facilitator superfamily (MFS) of transporter proteins and provide evidence that supports their split into domains and for multiple conformations within these families. We find that the ECOD and CATH provide the most extensive structural coverage of the PDB. ECOD and SCOPe have the most consistent domain boundary conditions, whereas CATH and SCOP2 both differ significantly.
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http://dx.doi.org/10.1021/acsomega.1c00950DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8223206PMC
June 2021

A combined RAD-Seq and WGS approach reveals the genomic basis of yellow color variation in bumble bee Bombus terrestris.

Sci Rep 2021 Apr 12;11(1):7996. Epub 2021 Apr 12.

Department of Biology, The Pennsylvania State University, 208 Mueller Labs, University Park, PA, USA.

Bumble bees exhibit exceptional diversity in their segmental body coloration largely as a result of mimicry. In this study we sought to discover genes involved in this variation through studying a lab-generated mutant in bumble bee Bombus terrestris, in which the typical black coloration of the pleuron, scutellum, and first metasomal tergite is replaced by yellow, a color variant also found in sister lineages to B. terrestris. Utilizing a combination of RAD-Seq and whole-genome re-sequencing, we localized the color-generating variant to a single SNP in the protein-coding sequence of transcription factor cut. This mutation generates an amino acid change that modifies the conformation of a coiled-coil structure outside DNA-binding domains. We found that all sequenced Hymenoptera, including sister lineages, possess the non-mutant allele, indicating different mechanisms are involved in the same color transition in nature. Cut is important for multiple facets of development, yet this mutation generated no noticeable external phenotypic effects outside of setal characteristics. Reproductive capacity was reduced, however, as queens were less likely to mate and produce female offspring, exhibiting behavior similar to that of workers. Our research implicates a novel developmental player in pigmentation, and potentially caste, thus contributing to a better understanding of the evolution of diversity in both of these processes.
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http://dx.doi.org/10.1038/s41598-021-87194-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8042027PMC
April 2021

Manipulation of IRE1-Dependent MAPK Signaling by a Vibrio Agonist-Antagonist Effector Pair.

mSystems 2021 Feb 9;6(1). Epub 2021 Feb 9.

Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas, USA

Diverse bacterial pathogens employ effector delivery systems to disrupt vital cellular processes in the host (N. M. Alto and K. Orth, Cold Spring Harbor Perspect Biol 4:a006114, 2012, https://doi.org/10.1101/cshperspect.a006114). The type III secretion system 1 of the marine pathogen utilizes the sequential action of four effectors to induce a rapid, proinflammatory cell death uniquely characterized by a prosurvival host transcriptional response (D. L. Burdette, M. L. Yarbrough, A Orvedahl, C. J. Gilpin, and K. Orth, Proc Natl Acad Sci USA 105:12497-12502, 2008, https://doi.org/10.1073/pnas.0802773105; N. J. De Nisco, M. Kanchwala, P. Li, J. Fernandez, C. Xing, and K. Orth, Sci Signal 10:eaa14501, 2017, https://doi.org/10.1126/scisignal.aal4501). Herein, we show that this prosurvival response is caused by the action of the channel-forming effector VopQ that targets the host V-ATPase, resulting in lysosomal deacidification and inhibition of lysosome-autophagosome fusion. Recent structural studies have shown how VopQ interacts with the V-ATPase and, while in the ER, a V-ATPase assembly intermediate can interact with VopQ, causing a disruption in membrane integrity. Additionally, we observed that VopQ-mediated disruption of the V-ATPase activates the IRE1 branch of the unfolded protein response (UPR), resulting in an IRE1-dependent activation of ERK1/2 MAPK signaling. We also find that this early VopQ-dependent induction of ERK1/2 phosphorylation is terminated by the VopS-mediated inhibitory AMPylation of Rho GTPase signaling. Since VopS dampens VopQ-induced IRE1-dependent ERK1/2 activation, we propose that IRE1 activates ERK1/2 phosphorylation at or above the level of Rho GTPases. This study illustrates how temporally induced effectors can work as in tandem as agonist/antagonist to manipulate host signaling and reveals new connections between V-ATPase function, UPR, and MAPK signaling. is a seafood-borne pathogen that encodes two type 3 secretion systems (T3SS). The first system, T3SS1, is thought to be maintained in all strains of to maintain survival in the environment, whereas the second system, T3SS2, is linked to clinical isolates and disease in humans. Here, we found that first system targets evolutionarily conserved signaling systems to manipulate host cells, eventually causing a rapid, orchestrated cells death within 3 h. We have found that the T3SS1 injects virulence factors that temporally manipulate host signaling. Within the first hour of infection, the effector VopQ acts first by activating host survival signals while diminishing the host cell apoptotic machinery. Less than an hour later, another effector, VopS, reverses activation and inhibition of these signaling systems, ultimately leading to death of the host cell. This work provides example of how pathogens have evolved to manipulate the interplay between T3SS effectors to regulate host signaling pathways.
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http://dx.doi.org/10.1128/mSystems.00872-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7883537PMC
February 2021

Genomics Reveals the Origins of Historical Specimens.

Mol Biol Evol 2021 05;38(5):2166-2176

Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, TX, USA.

Centuries of zoological studies have amassed billions of specimens in collections worldwide. Genomics of these specimens promises to reinvigorate biodiversity research. However, because DNA degrades with age in historical specimens, it is a challenge to obtain genomic data for them and analyze degraded genomes. We developed experimental and computational protocols to overcome these challenges and applied our methods to resolve a series of long-standing controversies involving a group of butterflies. We deduced the geographical origins of several historical specimens of uncertain provenance that are at the heart of these debates. Here, genomics tackles one of the greatest problems in zoology: countless old specimens that serve as irreplaceable embodiments of species concepts cannot be confidently assigned to extant species or population due to the lack of diagnostic morphological features and clear documentation of the collection locality. The ability to determine where they were collected will resolve many on-going disputes. More broadly, we show the utility of applying genomics to historical museum specimens to delineate the boundaries of species and populations, and to hypothesize about genotypic determinants of phenotypic traits.
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http://dx.doi.org/10.1093/molbev/msab013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8097301PMC
May 2021

A Fifth of the Protein World: Rossmann-like Proteins as an Evolutionarily Successful Structural unit.

J Mol Biol 2021 02 31;433(4):166788. Epub 2020 Dec 31.

Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, TX, United States; Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX, United States; Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX, United States. Electronic address:

The Rossmann-like fold is the most prevalent and diversified doubly-wound superfold of ancient evolutionary origin. Rossmann-like domains are present in a variety of metabolic enzymes and are capable of binding diverse ligands. Discerning evolutionary relationships among these domains is challenging because of their diverse functions and ancient origin. We defined a minimal Rossmann-like structural motif (RLM), identified RLM-containing domains among known 3D structures (20%) and classified them according to their homologous relationships. New classifications were incorporated into our Evolutionary Classification of protein Domains (ECOD) database. We defined 156 homology groups (H-groups), which were further clustered into 123 possible homology groups (X-groups). Our analysis revealed that RLM-containing proteins constitute approximately 15% of the human proteome. We found that disease-causing mutations are more frequent within RLM domains than within non-RLM domains of these proteins, highlighting the importance of RLM-containing proteins for human health.
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http://dx.doi.org/10.1016/j.jmb.2020.166788DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7870570PMC
February 2021

Structure, lipid scrambling activity and role in autophagosome formation of ATG9A.

Nat Struct Mol Biol 2020 12 26;27(12):1194-1201. Epub 2020 Oct 26.

Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA, USA.

De novo formation of the double-membrane compartment autophagosome is seeded by small vesicles carrying membrane protein autophagy-related 9 (ATG9), the function of which remains unknown. Here we find that ATG9A scrambles phospholipids of membranes in vitro. Cryo-EM structures of human ATG9A reveal a trimer with a solvated central pore, which is connected laterally to the cytosol through the cavity within each protomer. Similarities to ABC exporters suggest that ATG9A could be a transporter that uses the central pore to function. Moreover, molecular dynamics simulation suggests that the central pore opens laterally to accommodate lipid headgroups, thereby enabling lipids to flip. Mutations in the pore reduce scrambling activity and yield markedly smaller autophagosomes, indicating that lipid scrambling by ATG9A is essential for membrane expansion. We propose ATG9A acts as a membrane-embedded funnel to facilitate lipid flipping and to redistribute lipids added to the outer leaflet of ATG9 vesicles, thereby enabling growth into autophagosomes.
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http://dx.doi.org/10.1038/s41594-020-00520-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7718406PMC
December 2020

β-Strand-mediated interactions of protein domains.

Proteins 2020 11 11;88(11):1513-1527. Epub 2020 Jul 11.

Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

Protein domains exist by themselves or in combination with other domains to form complex multidomain proteins. Defining domain boundaries in proteins is essential for understanding their evolution and function but is not trivial. More specifically, partitioning domains that interact by forming a single β-sheet is known to be particularly troublesome for automatic structure-based domain decomposition pipelines. Here, we study edge-to-edge β-strand interactions between domains in a protein chain, to help define the boundaries for some more difficult cases where a single β-sheet spanning over two domains gives an appearance of one. We give a number of examples where β-strands belonging to a single β-sheet do not belong to a single domain and highlight the difficulties of automatic domain parsers on these examples. This work can be used as a baseline for defining domain boundaries in homologous proteins or proteins with similar domain interactions in the future.
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http://dx.doi.org/10.1002/prot.25970DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8018532PMC
November 2020

Mutation severity spectrum of rare alleles in the human genome is predictive of disease type.

PLoS Comput Biol 2020 05 15;16(5):e1007775. Epub 2020 May 15.

Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America.

The human genome harbors a variety of genetic variations. Single-nucleotide changes that alter amino acids in protein-coding regions are one of the major causes of human phenotypic variation and diseases. These single-amino acid variations (SAVs) are routinely found in whole genome and exome sequencing. Evaluating the functional impact of such genomic alterations is crucial for diagnosis of genetic disorders. We developed DeepSAV, a deep-learning convolutional neural network to differentiate disease-causing and benign SAVs based on a variety of protein sequence, structural and functional properties. Our method outperforms most stand-alone programs, and the version incorporating population and gene-level information (DeepSAV+PG) has similar predictive power as some of the best available. We transformed DeepSAV scores of rare SAVs in the human population into a quantity termed "mutation severity measure" for each human protein-coding gene. It reflects a gene's tolerance to deleterious missense mutations and serves as a useful tool to study gene-disease associations. Genes implicated in cancer, autism, and viral interaction are found by this measure as intolerant to mutations, while genes associated with a number of other diseases are scored as tolerant. Among known disease-associated genes, those that are mutation-intolerant are likely to function in development and signal transduction pathways, while those that are mutation-tolerant tend to encode metabolic and mitochondrial proteins.
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http://dx.doi.org/10.1371/journal.pcbi.1007775DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7255613PMC
May 2020

Schnyder corneal dystrophy-associated UBIAD1 is defective in MK-4 synthesis and resists autophagy-mediated degradation.

J Lipid Res 2020 05 18;61(5):746-757. Epub 2020 Mar 18.

Departments of Molecular Genetics,University of Texas Southwestern Medical Center, Dallas, TX 75390-9046. Electronic address:

The autosomal dominant disorder Schnyder corneal dystrophy (SCD) is caused by mutations in UbiA prenyltransferase domain-containing protein-1 (UBIAD1), which uses geranylgeranyl pyrophosphate (GGpp) to synthesize the vitamin K subtype menaquinone-4 (MK-4). SCD is characterized by opacification of the cornea, owing to aberrant build-up of cholesterol in the tissue. We previously discovered that sterols stimulate association of UBIAD1 with ER-localized HMG-CoA reductase, which catalyzes a rate-limiting step in the synthesis of cholesterol and nonsterol isoprenoids, including GGpp. Binding to UBIAD1 inhibits sterol-accelerated ER-associated degradation (ERAD) of reductase and permits continued synthesis of GGpp in cholesterol-replete cells. GGpp disrupts UBIAD1-reductase binding and thereby allows for maximal ERAD of reductase as well as ER-to-Golgi translocation of UBIAD1. SCD-associated UBIAD1 is refractory to GGpp-mediated dissociation from reductase and remains sequestered in the ER to inhibit ERAD. Here, we report development of a biochemical assay for UBIAD1-mediated synthesis of MK-4 in isolated membranes and intact cells. Using this assay, we compared enzymatic activity of WT UBIAD1 with that of SCD-associated variants. Our studies revealed that SCD-associated UBIAD1 exhibited reduced MK-4 synthetic activity, which may result from its reduced affinity for GGpp. Sequestration in the ER protects SCD-associated UBIAD1 from autophagy and allows intracellular accumulation of the mutant protein, which amplifies the inhibitory effect on reductase ERAD. These findings have important implications not only for the understanding of SCD etiology but also for the efficacy of cholesterol-lowering statin therapy, which becomes limited, in part, because of UBIAD1-mediated inhibition of reductase ERAD.
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http://dx.doi.org/10.1194/jlr.RA119000551DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7193952PMC
May 2020

Hyperactivation of TORC1 Drives Resistance to the Pan-HER Tyrosine Kinase Inhibitor Neratinib in HER2-Mutant Cancers.

Cancer Cell 2020 02 23;37(2):183-199.e5. Epub 2020 Jan 23.

Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, TX, USA; Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA. Electronic address:

We developed neratinib-resistant HER2-mutant cancer cells by gradual dose escalation. RNA sequencing identified TORC1 signaling as an actionable mechanism of drug resistance. Primary and acquired neratinib resistance in HER2-mutant breast cancer patient-derived xenografts (PDXs) was also associated with TORC1 hyperactivity. Genetic suppression of RAPTOR or RHEB ablated P-S6 and restored sensitivity to the tyrosine kinase inhibitor. The combination of the TORC1 inhibitor everolimus and neratinib potently arrested the growth of neratinib-resistant xenografts and organoids established from neratinib-resistant PDXs. RNA and whole-exome sequencing revealed RAS-mediated TORC1 activation in a subset of neratinib-resistant models. DNA sequencing of HER2-mutant tumors clinically refractory to neratinib, as well as circulating tumor DNA profiling of patients who progressed on neratinib, showed enrichment of genomic alterations that converge to activate the mTOR pathway.
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http://dx.doi.org/10.1016/j.ccell.2019.12.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7301608PMC
February 2020

A lytic polysaccharide monooxygenase-like protein functions in fungal copper import and meningitis.

Nat Chem Biol 2020 03 13;16(3):337-344. Epub 2020 Jan 13.

Department of Pharmacology and Cancer Biology, Duke University School of Medicine, Durham, NC, USA.

Infection by the fungal pathogen Cryptococcus neoformans causes lethal meningitis, primarily in immune-compromised individuals. Colonization of the brain by C. neoformans is dependent on copper (Cu) acquisition from the host, which drives critical virulence mechanisms. While C. neoformans Cu import and virulence are dependent on the Ctr1 and Ctr4 proteins, little is known concerning extracellular Cu ligands that participate in this process. We identified a C. neoformans gene, BIM1, that is strongly induced during Cu limitation and which encodes a protein related to lytic polysaccharide monooxygenases (LPMOs). Surprisingly, bim1 mutants are Cu deficient, and Bim1 function in Cu accumulation depends on Cu coordination and cell-surface association via a glycophosphatidyl inositol anchor. Bim1 participates in Cu uptake in concert with Ctr1 and expression of this pathway drives brain colonization in mouse infection models. These studies demonstrate a role for LPMO-like proteins as a critical factor for Cu acquisition in fungal meningitis.
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http://dx.doi.org/10.1038/s41589-019-0437-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7036007PMC
March 2020

RUVBL1/RUVBL2 ATPase Activity Drives PAQosome Maturation, DNA Replication and Radioresistance in Lung Cancer.

Cell Chem Biol 2020 01 26;27(1):105-121.e14. Epub 2019 Dec 26.

Eugene McDermott Center for Human Growth and Development, UT Southwestern Medical Center, Dallas, TX 75390, USA; Department of Pharmacology, UT Southwestern Medical Center, Dallas, TX 75390, USA; Harold C. Simmons Comprehensive Cancer Center, UT Southwestern Medical Center, Dallas, TX 75390, USA. Electronic address:

RUVBL1 and RUVBL2 (collectively RUVBL1/2) are essential AAA+ ATPases that function as co-chaperones and have been implicated in cancer. Here we investigated the molecular and phenotypic role of RUVBL1/2 ATPase activity in non-small cell lung cancer (NSCLC). We find that RUVBL1/2 are overexpressed in NSCLC patient tumors, with high expression associated with poor survival. Utilizing a specific inhibitor of RUVBL1/2 ATPase activity, we show that RUVBL1/2 ATPase activity is necessary for the maturation or dissociation of the PAQosome, a large RUVBL1/2-dependent multiprotein complex. We also show that RUVBL1/2 have roles in DNA replication, as inhibition of its ATPase activity can cause S-phase arrest, which culminates in cancer cell death via replication catastrophe. While in vivo pharmacological inhibition of RUVBL1/2 results in modest antitumor activity, it synergizes with radiation in NSCLC, but not normal cells, an attractive property for future preclinical development.
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http://dx.doi.org/10.1016/j.chembiol.2019.12.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8048778PMC
January 2020

Functional analysis of Rossmann-like domains reveals convergent evolution of topology and reaction pathways.

PLoS Comput Biol 2019 12 23;15(12):e1007569. Epub 2019 Dec 23.

Departments of Biophysics and Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America.

Rossmann folds are ancient, frequently diverged domains found in many biological reaction pathways where they have adapted for different functions. Consequently, discernment and classification of their homologous relations and function can be complicated. We define a minimal Rossmann-like structure motif (RLM) that corresponds for the common core of known Rossmann domains and use this motif to identify all RLM domains in the Protein Data Bank (PDB), thus finding they constitute about 20% of all known 3D structures. The Evolutionary Classification of protein structure Domains (ECOD) classifies RLM domains in a number of groups that lack evidence for homology (X-groups), which suggests that they could have evolved independently multiple times. Closely related, homologous RLM enzyme families can diverge to bind different ligands using similar binding sites and to catalyze different reactions. Conversely, non-homologous RLM domains can converge to catalyze the same reactions or to bind the same ligand with alternate binding modes. We discuss a special case of such convergent evolution that is relevant to the polypharmacology paradigm, wherein the same drug (methotrexate) binds to multiple non-homologous RLM drug targets with different topologies. Finally, assigning proteins with RLM domain to the Enzyme Commission classification suggest that RLM enzymes function mainly in metabolism (and comprise 38% of reference metabolic pathways) and are overrepresented in extant pathways that represent ancient biosynthetic routes such as nucleotide metabolism, energy metabolism, and metabolism of amino acids. In fact, RLM enzymes take part in five out of eight enzymatic reactions of the Wood-Ljungdahl metabolic pathway thought to be used by the last universal common ancestor (LUCA). The prevalence of RLM domains in this ancient metabolism might explain their wide distribution among enzymes.
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http://dx.doi.org/10.1371/journal.pcbi.1007569DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6957218PMC
December 2019

NK cell defects in X-linked pigmentary reticulate disorder.

JCI Insight 2019 11 1;4(21). Epub 2019 Nov 1.

Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

X-linked reticulate pigmentary disorder (XLPDR, Mendelian Inheritance in Man #301220) is a rare syndrome characterized by recurrent infections and sterile multiorgan inflammation. The syndrome is caused by an intronic mutation in POLA1, the gene encoding the catalytic subunit of DNA polymerase-α (Pol-α), which is responsible for Okazaki fragment synthesis during DNA replication. Reduced POLA1 expression in this condition triggers spontaneous type I interferon expression, which can be linked to the autoinflammatory manifestations of the disease. However, the history of recurrent infections in this syndrome is as yet unexplained. Here we report that patients with XLPDR have reduced NK cell cytotoxic activity and decreased numbers of NK cells, particularly differentiated, stage V, cells (CD3-CD56dim). This phenotype is reminiscent of hypomorphic mutations in MCM4, which encodes a component of the minichromosome maintenance (MCM) helicase complex that is functionally linked to Pol-α during the DNA replication process. We find that POLA1 deficiency leads to MCM4 depletion and that both can impair NK cell natural cytotoxicity and show that this is due to a defect in lytic granule polarization. Altogether, our study provides mechanistic connections between Pol-α and the MCM complex and demonstrates their relevance in NK cell function.
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http://dx.doi.org/10.1172/jci.insight.125688DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6948767PMC
November 2019

CASP13 target classification into tertiary structure prediction categories.

Proteins 2019 12 24;87(12):1021-1036. Epub 2019 Jul 24.

Departments of Biophysics and Biochemistry, Howard Hughes Medical Institute, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas.

Protein target structures for the Critical Assessment of Structure Prediction round 13 (CASP13) were split into evaluation units (EUs) based on their structural domains, the domain organization of available templates, and the performance of servers on whole targets compared to split target domains. Eighty targets were split into 112 EUs. The EUs were classified into categories suitable for assessment of high accuracy modeling (or template-based modeling [TBM]) and topology (or free modeling [FM]) based on target difficulty. Assignment into assessment categories considered the following criteria: (a) the evolutionary relationship of target domains to existing fold space as defined by the Evolutionary Classification of Protein Domains (ECOD) database; (b) the clustering of target domains using eight objective sequence, structure, and performance measures; and (c) the placement of target domains in a scatter plot of target difficulty against server performance used in the previous CASP. Generally, target domains with good server predictions had close template homologs and were classified as TBM. Alternately, targets with poor server predictions represent a mixture of fast evolving homologs, structure analogs, and new folds, and were classified as FM or FM/TBM overlap.
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http://dx.doi.org/10.1002/prot.25775DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6851465PMC
December 2019

Assessing predictions on fitness effects of missense variants in calmodulin.

Hum Mutat 2019 09 3;40(9):1463-1473. Epub 2019 Sep 3.

Departments of Biophysics and Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas.

This paper reports the evaluation of predictions for the "CALM1" challenge in the fifth round of the Critical Assessment of Genome Interpretation held in 2018. In the challenge, the participants were asked to predict effects on yeast growth caused by missense variants of human calmodulin, a highly conserved protein in eukaryotic cells sensing calcium concentration. The performance of predictors implementing different algorithms and methods is similar. Most predictors are able to identify the deleterious or tolerated variants with modest accuracy, with a baseline predictor based purely on sequence conservation slightly outperforming the submitted predictions. Nevertheless, we think that the accuracy of predictions remains far from satisfactory, and the field awaits substantial improvements. The most poorly predicted variants in this round surround functional CALM1 sites that bind calcium or peptide, which suggests that better incorporation of structural analysis may help improve predictions.
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http://dx.doi.org/10.1002/humu.23857DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6744288PMC
September 2019

Identification of residues critical for topology inversion of the transmembrane protein TM4SF20 through regulated alternative translocation.

J Biol Chem 2019 04 26;294(15):6054-6061. Epub 2019 Feb 26.

From the Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75390. Electronic address:

Adopting a proper topology is crucial for transmembrane proteins to perform their functions. We previously reported that ceramide regulates a transmembrane protein called TM4SF20 (transmembrane 4 L six family member 20) through topological inversion by altering the direction through which the protein is translocated across membranes during translation. This regulatory mechanism, denoted regulated alternative translocation (RAT), depends on a GN motif present in the first transmembrane helix of TM4SF20. Here, using site-directed mutagenesis, we show that Asn-26 in the motif is crucial for RAT of TM4SF20, as it cannot be replaced even by Gln. In contrast, Gly-22 in the motif could be substituted by other small residues such as Ala and Ser without affecting RAT of TM4SF20. We further demonstrate that the GN motif alone is insufficient to induce RAT of a transmembrane protein because TM4SF4, a relative of TM4SF20 that also contains the motif in the first transmembrane helix, did not undergo RAT. Using TM4SF40-TM4SF20 chimeras, we identified Pro-29 of TM4SF20 as another important element required for RAT of the protein. Substituting Pro-29 alone did not affect RAT of TM4SF20, whereas replacing Pro-29 together with either Leu-25 or Val-17 of TM4SF20 with the corresponding residues of TM4SF4 abolished RAT of TM4SF20. Because Val-17, Gly-22, Leu-25, Asn-26, and Pro-29 are predicted to reside along the same surface of the transmembrane helix, our results suggest that interactions with other proteins mediated by this surface during translocation may be critical for RAT of TM4SF20.
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http://dx.doi.org/10.1074/jbc.RA119.007681DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6463703PMC
April 2019

The effector RavD binds phosphatidylinositol-3-phosphate and helps suppress endolysosomal maturation of the -containing vacuole.

J Biol Chem 2019 04 7;294(16):6405-6415. Epub 2019 Feb 7.

From the Delaware Biotechnology Institute and

Upon phagocytosis into macrophages, the intracellular bacterial pathogen secretes effector proteins that manipulate host cell components, enabling it to evade lysosomal degradation. However, the bacterial proteins involved in this evasion are incompletely characterized. Here we show that the effector protein RavD targets host membrane compartments and contributes to the molecular mechanism the pathogen uses to prevent encounters with lysosomes. Protein-lipid binding assays revealed that RavD selectively binds phosphatidylinositol-3-phosphate (PI(3)P) We further determined that a C-terminal RavD region mediates the interaction with PI(3)P and that this interaction requires Arg-292. In transiently transfected mammalian cells, mCherry-RavD colocalized with the early endosome marker EGFP-Rab5 as well as the PI(3)P biosensor EGFP-2×FYVE. However, treatment with the phosphoinositide 3-kinase inhibitor wortmannin did not disrupt localization of mCherry-RavD to endosomal compartments, suggesting that RavD's interaction with PI(3)P is not necessary to anchor RavD to endosomal membranes. Using superresolution and immunogold transmission EM, we observed that, upon translocation into macrophages, RavD was retained onto the -containing vacuole and was also present on small vesicles adjacent to the vacuole. We also report that despite no detectable effects on intracellular growth of within macrophages or amebae, the lack of RavD significantly increased the number of vacuoles that accumulate the late endosome/lysosome marker LAMP-1 during macrophage infection. Together, our findings suggest that, although not required for intracellular replication of , RavD is a part of the molecular mechanism that steers the containing vacuole away from endolysosomal maturation pathways.
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http://dx.doi.org/10.1074/jbc.RA118.007086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6484141PMC
April 2019

Combined Blockade of Activating Mutations and ER Results in Synthetic Lethality of ER+/HER2 Mutant Breast Cancer.

Clin Cancer Res 2019 01 12;25(1):277-289. Epub 2018 Oct 12.

Department of Medicine, Vanderbilt-Ingram Cancer Center, Vanderbilt University Medical Center, Nashville, Tennessee.

Purpose: We examined the role of -activating mutations in endocrine therapy resistance in estrogen receptor positive (ER+) breast cancer.

Experimental Design: mutation frequency was determined from large genomic databases. Isogenic knock-in mutations in ER+ MCF7 cells and xenografts were used to investigate estrogen-independent growth. Structural analysis was used to determine the molecular interaction of with HER3. Small molecules and siRNAs were used to inhibit PI3Kα, TORC1, and HER3.

Results: Genomic data revealed a higher rate of mutations in metastatic versus primary ER+ tumors. MCF7 cells with isogenically incorporated kinase domain mutations exhibited resistance to estrogen deprivation and to fulvestrant both and , despite maintaining inhibition of ERα transcriptional activity. Addition of the irreversible HER2 tyrosine kinase inhibitor neratinib restored sensitivity to fulvestrant. HER2-mutant MCF7 cells expressed higher levels of p-HER3, p-AKT, and p-S6 than cells with wild-type HER2. Structural analysis of the HER2 variant implicated a more flexible active state, potentially allowing for enhanced dimerization with HER3. Treatment with a PI3Kα inhibitor, a TORC1 inhibitor or HER3 siRNA, but not a MEK inhibitor, restored sensitivity to fulvestrant and to estrogen deprivation. Inhibition of mutant HER2 or TORC1, when combined with fulvestrant, equipotently inhibited growth of MCF7/ xenografts, suggesting a role for TORC1 in antiestrogen resistance induced by mutations.

Conclusions: mutations hyperactivate the HER3/PI3K/AKT/mTOR axis, leading to antiestrogen resistance in ER+ breast cancer. Dual blockade of the HER2 and ER pathways is required for the treatment of ER+/HER2 mutant breast cancers.
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http://dx.doi.org/10.1158/1078-0432.CCR-18-1544DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6320312PMC
January 2019

Protein AMPylation by an Evolutionarily Conserved Pseudokinase.

Cell 2018 10 27;175(3):809-821.e19. Epub 2018 Sep 27.

Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA; Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA. Electronic address:

Approximately 10% of human protein kinases are believed to be inactive and named pseudokinases because they lack residues required for catalysis. Here, we show that the highly conserved pseudokinase selenoprotein-O (SelO) transfers AMP from ATP to Ser, Thr, and Tyr residues on protein substrates (AMPylation), uncovering a previously unrecognized activity for a member of the protein kinase superfamily. The crystal structure of a SelO homolog reveals a protein kinase-like fold with ATP flipped in the active site, thus providing a structural basis for catalysis. SelO pseudokinases localize to the mitochondria and AMPylate proteins involved in redox homeostasis. Consequently, SelO activity is necessary for the proper cellular response to oxidative stress. Our results suggest that AMPylation may be a more widespread post-translational modification than previously appreciated and that pseudokinases should be analyzed for alternative transferase activities.
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http://dx.doi.org/10.1016/j.cell.2018.08.046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6524645PMC
October 2018

FlyXCDB-A Resource for Drosophila Cell Surface and Secreted Proteins and Their Extracellular Domains.

J Mol Biol 2018 09 8;430(18 Pt B):3353-3411. Epub 2018 Jun 8.

Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Department of Biophysics and Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

Genomes of metazoan organisms possess a large number of genes encoding cell surface and secreted (CSS) proteins that carry out crucial functions in cell adhesion and communication, signal transduction, extracellular matrix establishment, nutrient digestion and uptake, immunity, and developmental processes. We developed the FlyXCDB database (http://prodata.swmed.edu/FlyXCDB) that provides a comprehensive resource to investigate extracellular (XC) domains in CSS proteins of Drosophila melanogaster, the most studied insect model organism in various aspects of animal biology. More than 300 Drosophila XC domains were discovered in Drosophila CSS proteins encoded by over 2500 genes through analyses of computational predictions of signal peptide, transmembrane (TM) segment, and GPI-anchor signal sequence, profile-based sequence similarity searches, gene ontology, and literature. These domains were classified into six classes mainly based on their molecular functions, including protein-protein interactions (class P), signaling molecules (class S), binding of non-protein molecules or groups (class B), enzyme homologs (class E), enzyme regulation and inhibition (class R), and unknown molecular function (class U). Main cellular functions such as cell adhesion, cell signaling, and extracellular matrix composition were described for the most abundant domains in each functional class. We assigned cell membrane topology categories (E, secreted; S, type I/III single-pass TM; T, type II single-pass TM; M, multi-pass TM; and G, GPI-anchored) to the products of genes with XC domains and investigated their regulation by mechanisms such as alternative splicing and stop codon readthrough.
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http://dx.doi.org/10.1016/j.jmb.2018.06.002DOI Listing
September 2018

Functional and evolutionary analysis of viral proteins containing a Rossmann-like fold.

Protein Sci 2018 08 13;27(8):1450-1463. Epub 2018 Jun 13.

Departments of Biophysics and Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas.

Viruses are the most abundant life form and infect practically all organisms. Consequently, these obligate parasites are a major cause of human suffering and economic loss. Rossmann-like fold is the most populated fold among α/β-folds in the Protein Data Bank and proteins containing Rossmann-like fold constitute 22% of all known proteins 3D structures. Thus, analysis of viral proteins containing Rossmann-like domains could provide an understanding of viral biology and evolution as well as could propose possible targets for antiviral therapy. We provide functional and evolutionary analysis of viral proteins containing a Rossmann-like fold found in the evolutionary classification of protein domains (ECOD) database developed in our lab. We identified 81 protein families of bacterial, archeal, and eukaryotic viruses in light of their evolution-based ECOD classification and Pfam taxonomy. We defined their functional significance using enzymatic EC number assignments as well as domain-level family annotations.
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http://dx.doi.org/10.1002/pro.3438DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6153405PMC
August 2018

Definition and classification of evaluation units for tertiary structure prediction in CASP12 facilitated through semi-automated metrics.

Proteins 2018 03 24;86 Suppl 1:16-26. Epub 2017 Oct 24.

Institute of Bioengineering, School of Life Sciences, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland.

For assessment purposes, CASP targets are split into evaluation units. We herein present the official definition of CASP12 evaluation units (EUs) and their classification into difficulty categories. Each target can be evaluated as one EU (the whole target) or/and several EUs (separate structural domains or groups of structural domains). The specific scenario for a target split is determined by the domain organization of available templates, the difference in server performance on separate domains versus combination of the domains, and visual inspection. In the end, 71 targets were split into 96 EUs. Classification of the EUs into difficulty categories was done semi-automatically with the assistance of metrics provided by the Prediction Center. These metrics account for sequence and structural similarities of the EUs to potential structural templates from the Protein Data Bank, and for the baseline performance of automated server predictions. The metrics readily separate the 96 EUs into 38 EUs that should be straightforward for template-based modeling (TBM) and 39 that are expected to be hard for homology modeling and are thus left for free modeling (FM). The remaining 19 borderline evaluation units were dubbed FM/TBM, and were inspected case by case. The article also overviews structural and evolutionary features of selected targets relevant to our accompanying article presenting the assessment of FM and FM/TBM predictions, and overviews structural features of the hardest evaluation units from the FM category. We finally suggest improvements for the EU definition and classification procedures.
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http://dx.doi.org/10.1002/prot.25403DOI Listing
March 2018

Assessing predictions of fitness effects of missense mutations in SUMO-conjugating enzyme UBE2I.

Hum Mutat 2017 09;38(9):1051-1063

Departments of Biophysics and Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas.

The exponential growth of genomic variants uncovered by next-generation sequencing necessitates efficient and accurate computational analyses to predict their functional effects. A number of computational methods have been developed for the task, but few unbiased comparisons of their performance are available. To fill the gap, The Critical Assessment of Genome Interpretation (CAGI) comprehensively assesses phenotypic predictions on newly collected experimental datasets. Here, we present the results of the SUMO conjugase challenge where participants were predicting functional effects of missense mutations in human SUMO-conjugating enzyme UBE2I. The performance of the predictors is similar to each other and is far from perfection. Evolutionary information from sequence alignments dominates the success: deleterious mutations at conserved positions and benign mutations at variable positions are accurately predicted. Prediction accuracy of other mutations remains unsatisfactory, and this fast-growing field of research is yet to learn the use of spatial structure information to improve the predictions significantly.
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http://dx.doi.org/10.1002/humu.23293DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5746193PMC
September 2017

Pet10p is a yeast perilipin that stabilizes lipid droplets and promotes their assembly.

J Cell Biol 2017 10 11;216(10):3199-3217. Epub 2017 Aug 11.

Department of Pharmacology, University of Texas Southwestern Medical School, Dallas, TX

Pet10p is a yeast lipid droplet protein of unknown function. We show that it binds specifically to and is stabilized by droplets containing triacylglycerol (TG). Droplets isolated from cells with a deletion strongly aggregate, appear fragile, and fuse in vivo when cells are cultured in oleic acid. Pet10p binds early to nascent droplets, and their rate of appearance is decreased in Moreover, Pet10p functionally interacts with the endoplasmic reticulum droplet assembly factors seipin and Fit2 to maintain proper droplet morphology. The activity of Dga1p, a diacylglycerol acyltransferase, and TG accumulation were both 30-35% lower in the absence of Pet10p. Pet10p contains a PAT domain, a defining property of perilipins, which was not previously known to exist in yeast. We propose that the core functions of Pet10p and other perilipins extend beyond protection from lipases and include the preservation of droplet integrity as well as collaboration with seipin and Fit2 in droplet assembly and maintenance.
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http://dx.doi.org/10.1083/jcb.201610013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5626530PMC
October 2017

Acute Hepatopancreatic Necrosis Disease-Causing Vibrio parahaemolyticus Strains Maintain an Antibacterial Type VI Secretion System with Versatile Effector Repertoires.

Appl Environ Microbiol 2017 07 16;83(13). Epub 2017 Jun 16.

Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas, USA

Acute hepatopancreatic necrosis disease (AHPND) is a newly emerging shrimp disease that has severely damaged the global shrimp industry. AHPND is caused by toxic strains of that have acquired a "selfish plasmid" encoding the deadly binary toxins PirA/PirB To better understand the repertoire of virulence factors in AHPND-causing , we conducted a comparative analysis using the genome sequences of the clinical strain RIMD2210633 and of environmental non-AHPND and toxic AHPND isolates of Interestingly, we found that all of the AHPND strains, but none of the non-AHPND strains, harbor the antibacterial type VI secretion system 1 (T6SS1), which we previously identified and characterized in the clinical isolate RIMD2210633. This finding suggests that the acquisition of this T6SS might confer to AHPND-causing a fitness advantage over competing bacteria and facilitate shrimp infection. Additionally, we found highly dynamic effector loci in the T6SS1 of AHPND-causing strains, leading to diverse effector repertoires. Our discovery provides novel insights into AHPND-causing pathogens and reveals a potential target for disease control. Acute hepatopancreatic necrosis disease (AHPND) is a serious disease that has caused severe damage and significant financial losses to the global shrimp industry. To better understand and prevent this shrimp disease, it is essential to thoroughly characterize its causative agent, Although the plasmid-encoded binary toxins PirA/PirB have been shown to be the primary cause of AHPND, it remains unknown whether other virulent factors are commonly present in and might play important roles during shrimp infection. Here, we analyzed the genome sequences of clinical, non-AHPND, and AHPND strains to characterize their repertoires of key virulence determinants. Our studies reveal that an antibacterial type VI secretion system is associated with the AHPND strains and differentiates them from non-AHPND strains, similar to what was seen with the PirA/PirB toxins. We propose that T6SS1 provides a selective advantage during shrimp infections.
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http://dx.doi.org/10.1128/AEM.00737-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5478980PMC
July 2017
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