Publications by authors named "John K Everett"

53 Publications

Lentiviral globin gene therapy with reduced-intensity conditioning in adults with β-thalassemia: a phase 1 trial.

Nat Med 2022 Jan 3. Epub 2022 Jan 3.

Department of Microbiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA.

β-Thalassemias are inherited anemias that are caused by the absent or insufficient production of the β chain of hemoglobin. Here we report 6-8-year follow-up of four adult patients with transfusion-dependent β-thalassemia who were infused with autologous CD34 cells transduced with the TNS9.3.55 lentiviral globin vector after reduced-intensity conditioning (RIC) in a phase 1 clinical trial ( NCT01639690) . Patients were monitored for insertional mutagenesis and the generation of a replication-competent lentivirus (safety and tolerability of the infusion product after RIC-primary endpoint) and engraftment of genetically modified autologous CD34 cells, expression of the transduced β-globin gene and post-transplant transfusion requirements (efficacy-secondary endpoint). No unexpected safety issues occurred during conditioning and cell product infusion. Hematopoietic gene marking was very stable but low, reducing transfusion requirements in two patients, albeit not achieving transfusion independence. Our findings suggest that non-myeloablative conditioning can achieve durable stem cell engraftment but underscore a minimum CD34 cell transduction requirement for effective therapy. Moderate clonal expansions were associated with integrations near cancer-related genes, suggestive of non-erythroid activity of globin vectors in stem/progenitor cells. These correlative findings highlight the necessity of cautiously monitoring patients harboring globin vectors.
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http://dx.doi.org/10.1038/s41591-021-01554-9DOI Listing
January 2022

BET bromodomain protein inhibition reverses chimeric antigen receptor extinction and reinvigorates exhausted T cells in chronic lymphocytic leukemia.

J Clin Invest 2021 08;131(16)

Center for Cellular Immunotherapies.

Chimeric antigen receptor (CAR) T cells have induced remarkable antitumor responses in B cell malignancies. Some patients do not respond because of T cell deficiencies that hamper the expansion, persistence, and effector function of these cells. We used longitudinal immune profiling to identify phenotypic and pharmacodynamic changes in CD19-directed CAR T cells in patients with chronic lymphocytic leukemia (CLL). CAR expression maintenance was also investigated because this can affect response durability. CAR T cell failure was accompanied by preexisting T cell-intrinsic defects or dysfunction acquired after infusion. In a small subset of patients, CAR silencing was observed coincident with leukemia relapse. Using a small molecule inhibitor, we demonstrated that the bromodomain and extra-terminal (BET) family of chromatin adapters plays a role in downregulating CAR expression. BET protein blockade also ameliorated CAR T cell exhaustion as manifested by inhibitory receptor reduction, enhanced metabolic fitness, increased proliferative capacity, and enriched transcriptomic signatures of T cell reinvigoration. BET inhibition decreased levels of the TET2 methylcytosine dioxygenase, and forced expression of the TET2 catalytic domain eliminated the potency-enhancing effects of BET protein targeting in CAR T cells, providing a mechanism linking BET proteins and T cell dysfunction. Thus, modulating BET epigenetic readers may improve the efficacy of cell-based immunotherapies.
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http://dx.doi.org/10.1172/JCI145459DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8363276PMC
August 2021

NPM-ALK-Induced Reprogramming of Mature TCR-Stimulated T Cells Results in Dedifferentiation and Malignant Transformation.

Cancer Res 2021 06 22;81(12):3241-3254. Epub 2021 Feb 22.

Department of Microbiology, University of Pennsylvania, Philadelphia, Pennsylvania.

Fusion genes including NPM-ALK can promote T-cell transformation, but the signals required to drive a healthy T cell to become malignant remain undefined. In this study, we introduce NPM-ALK into primary human T cells and demonstrate induction of the epithelial-to-mesenchymal transition (EMT) program, attenuation of most T-cell effector programs, reemergence of an immature epigenomic profile, and dynamic regulation of c-Myc, E2F, and PI3K/mTOR signaling pathways early during transformation. A mutant of NPM-ALK failed to bind several signaling complexes including GRB2/SOS, SHC1, SHC4, and UBASH3B and was unable to transform T cells. Finally, T-cell receptor (TCR)-generated signals were required to achieve T-cell transformation, explaining how healthy individuals can harbor T cells with NPM-ALK translocations. These findings describe the fundamental mechanisms of NPM-ALK-mediated oncogenesis and may serve as a model to better understand factors that regulate tumor formation. SIGNIFICANCE: This investigation into malignant transformation of T cells uncovers a requirement for TCR triggering, elucidates integral signaling complexes nucleated by NPM-ALK, and delineates dynamic transcriptional changes as a T cell transforms..
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http://dx.doi.org/10.1158/0008-5472.CAN-20-2297DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8260452PMC
June 2021

Antigen-driven clonal selection shapes the persistence of HIV-1-infected CD4+ T cells in vivo.

J Clin Invest 2021 02;131(3)

Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

Clonal expansion of infected CD4+ T cells is a major mechanism of HIV-1 persistence and a barrier to achieving a cure. Potential causes are homeostatic proliferation, effects of HIV-1 integration, and interaction with antigens. Here, we show that it is possible to link antigen responsiveness, the full proviral sequence, the integration site, and the T cell receptor β-chain (TCRβ) sequence to examine the role of recurrent antigenic exposure in maintaining the HIV-1 reservoir. We isolated CMV- and Gag-responding CD4+ T cells from 10 treated individuals. Proviral populations in CMV-responding cells were dominated by large clones, including clones harboring replication-competent proviruses. TCRβ repertoires showed high clonality driven by converging adaptive responses. Although some proviruses were in genes linked to HIV-1 persistence (BACH2, STAT5B, MKL1), the proliferation of infected cells under antigenic stimulation occurred regardless of the site of integration. Paired TCRβ and integration site analysis showed that infection could occur early or late in the course of a clone's response to antigen and could generate infected cell populations too large to be explained solely by homeostatic proliferation. Together, these findings implicate antigen-driven clonal selection as a major factor in HIV-1 persistence, a finding that will be a difficult challenge to eradication efforts.
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http://dx.doi.org/10.1172/JCI145254DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7843227PMC
February 2021

A long-term study of AAV gene therapy in dogs with hemophilia A identifies clonal expansions of transduced liver cells.

Nat Biotechnol 2021 01 16;39(1):47-55. Epub 2020 Nov 16.

The Raymond G. Perelman Center for Cellular and Molecular Therapeutics, Children's Hospital of Philadelphia, Philadelphia, PA, USA.

Nine dogs with hemophilia A were treated with adeno-associated viral (AAV) gene therapy and followed for up to 10 years. Administration of AAV8 or AAV9 vectors expressing canine factor VIII (AAV-cFVIII) corrected the FVIII deficiency to 1.9-11.3% of normal FVIII levels. In two of nine dogs, levels of FVIII activity increased gradually starting about 4 years after treatment. None of the dogs showed evidence of tumors or altered liver function. Analysis of integration sites in liver samples from six treated dogs identified 1,741 unique AAV integration events in genomic DNA and expanded cell clones in five dogs, with 44% of the integrations near genes involved in cell growth. All recovered integrated vectors were partially deleted and/or rearranged. Our data suggest that the increase in FVIII protein expression in two dogs may have been due to clonal expansion of cells harboring integrated vectors. These results support the clinical development of liver-directed AAV gene therapy for hemophilia A, while emphasizing the importance of long-term monitoring for potential genotoxicity.
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http://dx.doi.org/10.1038/s41587-020-0741-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7855056PMC
January 2021

Assessment of HIV-1 integration in tissues and subsets across infection stages.

JCI Insight 2020 10 15;5(20). Epub 2020 Oct 15.

Department of Microbiology and.

The integration of HIV DNA into the host genome contributes to lifelong infection in most individuals. Few studies have examined integration in lymphoid tissue, where HIV predominantly persists before and after antiretroviral treatment (ART). Of particular interest is whether integration site distributions differ between infection stages with paired blood and tissue comparisons. Here, we profiled HIV integration site distributions in sorted memory, tissue-resident, and/or follicular helper CD4+ T cell subsets from paired blood and lymphoid tissue samples from acute, chronic, and ART-treated individuals. We observed minor differences in the frequency of nonintronic and nondistal intergenic sites, varying with tissue and residency phenotypes during ART. Genomic and epigenetic annotations were generally similar. Clonal expansion of cells marked by identical integration sites was detected, with increased detection in chronic and ART-treated individuals. However, overlap between or within CD4+ T cell subsets or tissue compartments was only observed in 8 unique sites of the 3540 sites studied. Together, these findings suggest that shared integration sites between blood and tissue may, depending on the tissue site, be the exception rather than the rule and indicate that additional studies are necessary to fully understand the heterogeneity of tissue-sequestered HIV reservoirs.
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http://dx.doi.org/10.1172/jci.insight.139783DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7605534PMC
October 2020

Clonal tracking in gene therapy patients reveals a diversity of human hematopoietic differentiation programs.

Blood 2020 04;135(15):1219-1231

Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, PA.

In gene therapy with human hematopoietic stem and progenitor cells (HSPCs), each gene-corrected cell and its progeny are marked in a unique way by the integrating vector. This feature enables lineages to be tracked by sampling blood cells and using DNA sequencing to identify the vector integration sites. Here, we studied 5 cell lineages (granulocytes, monocytes, T cells, B cells, and natural killer cells) in patients having undergone HSPC gene therapy for Wiskott-Aldrich syndrome or β hemoglobinopathies. We found that the estimated minimum number of active, repopulating HSPCs (which ranged from 2000 to 50 000) was correlated with the number of HSPCs per kilogram infused. We sought to quantify the lineage output and dynamics of gene-modified clones; this is usually challenging because of sparse sampling of the various cell types during the analytical procedure, contamination during cell isolation, and different levels of vector marking in the various lineages. We therefore measured the residual contamination and corrected our statistical models accordingly to provide a rigorous analysis of the HSPC lineage output. A cluster analysis of the HSPC lineage output highlighted the existence of several stable, distinct differentiation programs, including myeloid-dominant, lymphoid-dominant, and balanced cell subsets. Our study evidenced the heterogeneous nature of the cell lineage output from HSPCs and provided methods for analyzing these complex data.
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http://dx.doi.org/10.1182/blood.2019002350DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7146019PMC
April 2020

Lentiviral gene therapy for X-linked chronic granulomatous disease.

Nat Med 2020 02 27;26(2):200-206. Epub 2020 Jan 27.

Great Ormond Street Institute of Child Health and Great Ormond Street Hospital NHS Foundation Trust, London, UK.

Chronic granulomatous disease (CGD) is a rare inherited disorder of phagocytic cells. We report the initial results of nine severely affected X-linked CGD (X-CGD) patients who received ex vivo autologous CD34 hematopoietic stem and progenitor cell-based lentiviral gene therapy following myeloablative conditioning in first-in-human studies (trial registry nos. NCT02234934 and NCT01855685). The primary objectives were to assess the safety and evaluate the efficacy and stability of biochemical and functional reconstitution in the progeny of engrafted cells at 12 months. The secondary objectives included the evaluation of augmented immunity against bacterial and fungal infection, as well as assessment of hematopoietic stem cell transduction and engraftment. Two enrolled patients died within 3 months of treatment from pre-existing comorbidities. At 12 months, six of the seven surviving patients demonstrated stable vector copy numbers (0.4-1.8 copies per neutrophil) and the persistence of 16-46% oxidase-positive neutrophils. There was no molecular evidence of either clonal dysregulation or transgene silencing. Surviving patients have had no new CGD-related infections, and six have been able to discontinue CGD-related antibiotic prophylaxis. The primary objective was met in six of the nine patients at 12 months follow-up, suggesting that autologous gene therapy is a promising approach for CGD patients.
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http://dx.doi.org/10.1038/s41591-019-0735-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7115833PMC
February 2020

CD19-targeting CAR T cell immunotherapy outcomes correlate with genomic modification by vector integration.

J Clin Invest 2020 02;130(2):673-685

Department of Microbiology.

Chimeric antigen receptor-engineered T cells targeting CD19 (CART19) provide an effective treatment for pediatric acute lymphoblastic leukemia but are less effective for chronic lymphocytic leukemia (CLL), focusing attention on improving efficacy. CART19 harbor an engineered receptor, which is delivered through lentiviral vector integration, thereby marking cell lineages and modifying the cellular genome by insertional mutagenesis. We recently reported that vector integration within the host TET2 gene was associated with CLL remission. Here, we investigated clonal population structure and therapeutic outcomes in another 39 patients by high-throughput sequencing of vector-integration sites. Genes at integration sites enriched in responders were commonly found in cell-signaling and chromatin modification pathways, suggesting that insertional mutagenesis in these genes promoted therapeutic T cell proliferation. We also developed a multivariate model based on integration-site distributions and found that data from preinfusion products forecasted response in CLL successfully in discovery and validation cohorts and, in day 28 samples, reported responders to CLL therapy with high accuracy. These data clarify how insertional mutagenesis can modulate cell proliferation in CART19 therapy and how data on integration-site distributions can be linked to treatment outcomes.
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http://dx.doi.org/10.1172/JCI130144DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6994131PMC
February 2020

An ELISA-Based Screening Platform for Ligand-Receptor Discovery.

Methods Enzymol 2019 28;615:453-475. Epub 2018 Dec 28.

Child Health Institute of New Jersey, Robert Wood Johnson Medical School, Rutgers, The State University of New Jersey, New Brunswick, NJ, United States; Department of Neuroscience and Cell Biology, Robert Wood Johnson Medical School, Rutgers, The State University of New Jersey, New Brunswick, NJ, United States; Department of Pediatrics, Robert Wood Johnson Medical School, Rutgers, The State University of New Jersey, New Brunswick, NJ, United States. Electronic address:

Cell surface molecules are important for development and function of multicellular organisms. Although several methods are available to identify ligand-receptor pairs, ELISA-based methods are particularly amenable to high-throughput screens. ELISA-based methods have high sensitivity and low false-positive rates for detecting protein-protein interaction (PPI) complexes. Here, we provide a detailed protocol for a 384-well ELISA-based PPI screening protocol for the identification of novel cell surface ligand-receptor interactions, together with considerations for validation of PPIs by biophysical methods. This PPI screen has been developed and tested for discovery of novel ligand-receptor pairs between human synaptic adhesion proteins, believed to play crucial roles in many steps of neurodevelopment, from neuronal maturation, to axon guidance, synapse connectivity, and pruning.
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http://dx.doi.org/10.1016/bs.mie.2018.10.001DOI Listing
November 2019

T cell dynamics and response of the microbiota after gene therapy to treat X-linked severe combined immunodeficiency.

Genome Med 2018 09 28;10(1):70. Epub 2018 Sep 28.

Department of Microbiology, University of Pennsylvania School of Medicine, 3610 Hamilton Walk, Philadelphia, PA, 19104-6076, USA.

Background: Mutation of the IL2RG gene results in a form of severe combined immune deficiency (SCID-X1), which has been treated successfully with hematopoietic stem cell gene therapy. SCID-X1 gene therapy results in reconstitution of the previously lacking T cell compartment, allowing analysis of the roles of T cell immunity in humans by comparing before and after gene correction.

Methods: Here we interrogate T cell reconstitution using four forms of high throughput analysis. (1) Estimation of the numbers of transduced progenitor cells by monitoring unique positions of integration of the therapeutic gene transfer vector. (2) Estimation of T cell population structure by sequencing of the recombined T cell receptor (TCR) beta locus. (3) Metagenomic analysis of microbial populations in oropharyngeal, nasopharyngeal, and gut samples. (4) Metagenomic analysis of viral populations in gut samples.

Results: Comparison of progenitor and mature T cell populations allowed estimation of a minimum number of cell divisions needed to generate the observed populations. Analysis of microbial populations showed the effects of immune reconstitution, including normalization of gut microbiota and clearance of viral infections. Metagenomic analysis revealed enrichment of genes for antibiotic resistance in gene-corrected subjects relative to healthy controls, likely a result of higher healthcare exposure.

Conclusions: This multi-omic approach enables the characterization of multiple effects of SCID-X1 gene therapy, including T cell repertoire reconstitution, estimation of numbers of cell divisions between progenitors and daughter T cells, normalization of the microbiome, clearance of microbial pathogens, and modulations in antibiotic resistance gene levels. Together, these results quantify several aspects of the long-term efficacy of gene therapy for SCID-X1. This study includes data from ClinicalTrials.gov numbers NCT01410019, NCT01175239, and NCT01129544.
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http://dx.doi.org/10.1186/s13073-018-0580-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6161392PMC
September 2018

Disruption of TET2 promotes the therapeutic efficacy of CD19-targeted T cells.

Nature 2018 06 30;558(7709):307-312. Epub 2018 May 30.

Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.

Cancer immunotherapy based on genetically redirecting T cells has been used successfully to treat B cell malignancies. In this strategy, the T cell genome is modified by integration of viral vectors or transposons encoding chimaeric antigen receptors (CARs) that direct tumour cell killing. However, this approach is often limited by the extent of expansion and persistence of CAR T cells. Here we report mechanistic insights from studies of a patient with chronic lymphocytic leukaemia treated with CAR T cells targeting the CD19 protein. Following infusion of CAR T cells, anti-tumour activity was evident in the peripheral blood, lymph nodes and bone marrow; this activity was accompanied by complete remission. Unexpectedly, at the peak of the response, 94% of CAR T cells originated from a single clone in which lentiviral vector-mediated insertion of the CAR transgene disrupted the methylcytosine dioxygenase TET2 gene. Further analysis revealed a hypomorphic mutation in this patient's second TET2 allele. TET2-disrupted CAR T cells exhibited an epigenetic profile consistent with altered T cell differentiation and, at the peak of expansion, displayed a central memory phenotype. Experimental knockdown of TET2 recapitulated the potency-enhancing effect of TET2 dysfunction in this patient's CAR T cells. These findings suggest that the progeny of a single CAR T cell induced leukaemia remission and that TET2 modification may be useful for improving immunotherapies.
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http://dx.doi.org/10.1038/s41586-018-0178-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6320248PMC
June 2018

INSPIIRED: A Pipeline for Quantitative Analysis of Sites of New DNA Integration in Cellular Genomes.

Mol Ther Methods Clin Dev 2017 Mar 18;4:39-49. Epub 2016 Dec 18.

Department of Microbiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104-6076, USA.

Integration of new DNA into cellular genomes mediates replication of retroviruses and transposons; integration reactions have also been adapted for use in human gene therapy. Tracking the distributions of integration sites is important to characterize populations of transduced cells and to monitor potential outgrow of pathogenic cell clones. Here, we describe a pipeline for quantitative analysis of integration site distributions named INSPIIRED (integration site pipeline for paired-end reads). We describe optimized biochemical steps for site isolation using Illumina paired-end sequencing, including new technology for suppressing recovery of unwanted contaminants, then software for alignment, quality control, and management of integration site sequences. During library preparation, DNAs are broken by sonication, so that after ligation-mediated PCR the number of ligation junction sites can be used to infer abundance of gene-modified cells. We generated integration sites of known positions in silico, and we describe optimization of sample processing parameters refined by comparison to truth. We also present a novel graph-theory-based method for quantifying integration sites in repeated sequences, and we characterize the consequences using synthetic and experimental data. In an accompanying paper, we describe an additional set of statistical tools for data analysis and visualization. Software is available at https://github.com/BushmanLab/INSPIIRED.
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http://dx.doi.org/10.1016/j.omtm.2016.11.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5363316PMC
March 2017

INSPIIRED: Quantification and Visualization Tools for Analyzing Integration Site Distributions.

Mol Ther Methods Clin Dev 2017 Mar 18;4:17-26. Epub 2016 Dec 18.

Department of Microbiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104-6076, USA.

Analysis of sites of newly integrated DNA in cellular genomes is important to several fields, but methods for analyzing and visualizing these datasets are still under development. Here, we describe tools for data analysis and visualization that take as input integration site data from our INSPIIRED pipeline. Paired-end sequencing allows inference of the numbers of transduced cells as well as the distributions of integration sites in target genomes. We present interactive heatmaps that allow comparison of distributions of integration sites to genomic features and that support numerous user-defined statistical tests. To summarize integration site data from human gene therapy samples, we developed a reproducible report format that catalogs sample population structure, longitudinal dynamics, and integration frequency near cancer-associated genes. We also introduce a novel summary statistic, the UC50 (unique cell progenitors contributing the most expanded 50% of progeny cell clones), which provides a single number summarizing possible clonal expansion. Using these tools, we characterize ongoing longitudinal characterization of a patient from the first trial to treat severe combined immunodeficiency-X1 (SCID-X1), showing successful reconstitution for 15 years accompanied by persistence of a cell clone with an integration site near the cancer-associated gene CCND2. Software is available at https://github.com/BushmanLab/INSPIIRED.
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http://dx.doi.org/10.1016/j.omtm.2016.11.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5363318PMC
March 2017

Accounting for Protein Subcellular Localization: A Compartmental Map of the Rat Liver Proteome.

Mol Cell Proteomics 2017 02 6;16(2):194-212. Epub 2016 Dec 6.

¶Center for Advanced Biotechnology and Medicine, Rutgers Biomedical and Health Sciences, 679 Hoes Lane West, Piscataway, New Jersey 08854;

Accurate knowledge of the intracellular location of proteins is important for numerous areas of biomedical research including assessing fidelity of putative protein-protein interactions, modeling cellular processes at a system-wide level and investigating metabolic and disease pathways. Many proteins have not been localized, or have been incompletely localized, partly because most studies do not account for entire subcellular distribution. Thus, proteins are frequently assigned to one organelle whereas a significant fraction may reside elsewhere. As a step toward a comprehensive cellular map, we used subcellular fractionation with classic balance sheet analysis and isobaric labeling/quantitative mass spectrometry to assign locations to >6000 rat liver proteins. We provide quantitative data and error estimates describing the distribution of each protein among the eight major cellular compartments: nucleus, mitochondria, lysosomes, peroxisomes, endoplasmic reticulum, Golgi, plasma membrane and cytosol. Accounting for total intracellular distribution improves quality of organelle assignments and assigns proteins with multiple locations. Protein assignments and supporting data are available online through the Prolocate website (http://prolocate.cabm.rutgers.edu). As an example of the utility of this data set, we have used organelle assignments to help analyze whole exome sequencing data from an infant dying at 6 months of age from a suspected neurodegenerative lysosomal storage disorder of unknown etiology. Sequencing data was prioritized using lists of lysosomal proteins comprising well-established residents of this organelle as well as novel candidates identified in this study. The latter included copper transporter 1, encoded by SLC31A1, which we localized to both the plasma membrane and lysosome. The patient harbors two predicted loss of function mutations in SLC31A1, suggesting that this may represent a heretofore undescribed recessive lysosomal storage disease gene.
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http://dx.doi.org/10.1074/mcp.M116.064527DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5294208PMC
February 2017

Introduction of a polar core into the de novo designed protein Top7.

Protein Sci 2016 07 7;25(7):1299-307. Epub 2016 Mar 7.

Department of Biochemistry, University of Washington, Seattle, Washington, 98195.

Design of polar interactions is a current challenge for protein design. The de novo designed protein Top7, like almost all designed proteins, has an entirely nonpolar core. Here we describe the replacing of a sizable fraction (5 residues) of this core with a designed polar hydrogen bond network. The polar core design is expressed at high levels in E. coli, has a folding free energy of 10 kcal/mol, and retains the multiphasic folding kinetics of the original Top7. The NMR structure of the design shows that conformations of three of the five residues, and the designed hydrogen bonds between them, are very close to those in the design model. The remaining two residues, which are more solvent exposed, sample a wide range of conformations in the NMR ensemble. These results show that hydrogen bond networks can be designed in protein cores, but also highlight challenges that need to be overcome when there is competition with solvent.
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http://dx.doi.org/10.1002/pro.2899DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4918430PMC
July 2016

The detection and subsequent volume optimization of biological nanocrystals.

Struct Dyn 2015 Jul 15;2(4):041710. Epub 2015 May 15.

Department of Molecular Biology and Biochemistry, Center for Advanced Biotechnology and Medicine, and Northeast Structural Genomics Consortium, Rutgers, The State University of New Jersey , 679 Hoes Lane, Piscataway, New Jersey 08854-8021, USA.

Identifying and then optimizing initial crystallization conditions is a prerequisite for macromolecular structure determination by crystallography. Improved technologies enable data collection on crystals that are difficult if not impossible to detect using visible imaging. The application of second-order nonlinear imaging of chiral crystals and ultraviolet two-photon excited fluorescence detection is shown to be applicable in a high-throughput manner to rapidly verify the presence of nanocrystals in crystallization screening conditions. It is noted that the nanocrystals are rarely seen without also producing microcrystals from other chemical conditions. A crystal volume optimization method is described and associated with a phase diagram for crystallization.
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http://dx.doi.org/10.1063/1.4921199DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4711624PMC
July 2015

Codon influence on protein expression in E. coli correlates with mRNA levels.

Nature 2016 Jan 13;529(7586):358-363. Epub 2016 Jan 13.

Department of Biological Sciences, 702A Fairchild Center, MC2434, Columbia University, New York, NY 10027, USA.

Degeneracy in the genetic code, which enables a single protein to be encoded by a multitude of synonymous gene sequences, has an important role in regulating protein expression, but substantial uncertainty exists concerning the details of this phenomenon. Here we analyse the sequence features influencing protein expression levels in 6,348 experiments using bacteriophage T7 polymerase to synthesize messenger RNA in Escherichia coli. Logistic regression yields a new codon-influence metric that correlates only weakly with genomic codon-usage frequency, but strongly with global physiological protein concentrations and also mRNA concentrations and lifetimes in vivo. Overall, the codon content influences protein expression more strongly than mRNA-folding parameters, although the latter dominate in the initial ~16 codons. Genes redesigned based on our analyses are transcribed with unaltered efficiency but translated with higher efficiency in vitro. The less efficiently translated native sequences show greatly reduced mRNA levels in vivo. Our results suggest that codon content modulates a kinetic competition between protein elongation and mRNA degradation that is a central feature of the physiology and also possibly the regulation of translation in E. coli.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5054687PMC
http://dx.doi.org/10.1038/nature16509DOI Listing
January 2016

A community resource of experimental data for NMR / X-ray crystal structure pairs.

Protein Sci 2016 Jan 22;25(1):30-45. Epub 2015 Sep 22.

Cancer Genomics & Proteomics, Department of Medical Biophysics, Ontario Cancer Institute, and Northeast Structural Genomics Consortium, University of Toronto, Toronto, Ontario, M5G 1L7, Canada.

We have developed an online NMR / X-ray Structure Pair Data Repository. The NIGMS Protein Structure Initiative (PSI) has provided many valuable reagents, 3D structures, and technologies for structural biology. The Northeast Structural Genomics Consortium was one of several PSI centers. NESG used both X-ray crystallography and NMR spectroscopy for protein structure determination. A key goal of the PSI was to provide experimental structures for at least one representative of each of hundreds of targeted protein domain families. In some cases, structures for identical (or nearly identical) constructs were determined by both NMR and X-ray crystallography. NMR spectroscopy and X-ray diffraction data for 41 of these "NMR / X-ray" structure pairs determined using conventional triple-resonance NMR methods with extensive sidechain resonance assignments have been organized in an online NMR / X-ray Structure Pair Data Repository. In addition, several NMR data sets for perdeuterated, methyl-protonated protein samples are included in this repository. As an example of the utility of this repository, these data were used to revisit questions about the precision and accuracy of protein NMR structures first outlined by Levy and coworkers several years ago (Andrec et al., Proteins 2007;69:449-465). These results demonstrate that the agreement between NMR and X-ray crystal structures is improved using modern methods of protein NMR spectroscopy. The NMR / X-ray Structure Pair Data Repository will provide a valuable resource for new computational NMR methods development.
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http://dx.doi.org/10.1002/pro.2774DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4815321PMC
January 2016

Mitochondrial COQ9 is a lipid-binding protein that associates with COQ7 to enable coenzyme Q biosynthesis.

Proc Natl Acad Sci U S A 2014 Nov 22;111(44):E4697-705. Epub 2014 Oct 22.

Departments of Biochemistry, Mitochondrial Protein Partnership, University of Wisconsin-Madison, Madison, WI 53706;

Coenzyme Q (CoQ) is an isoprenylated quinone that is essential for cellular respiration and is synthesized in mitochondria by the combined action of at least nine proteins (COQ1-9). Although most COQ proteins are known to catalyze modifications to CoQ precursors, the biochemical role of COQ9 remains unclear. Here, we report that a disease-related COQ9 mutation leads to extensive disruption of the CoQ protein biosynthetic complex in a mouse model, and that COQ9 specifically interacts with COQ7 through a series of conserved residues. Toward understanding how COQ9 can perform these functions, we solved the crystal structure of Homo sapiens COQ9 at 2.4 Å. Unexpectedly, our structure reveals that COQ9 has structural homology to the TFR family of bacterial transcriptional regulators, but that it adopts an atypical TFR dimer orientation and is not predicted to bind DNA. Our structure also reveals a lipid-binding site, and mass spectrometry-based analyses of purified COQ9 demonstrate that it associates with multiple lipid species, including CoQ itself. The conserved COQ9 residues necessary for its interaction with COQ7 comprise a surface patch around the lipid-binding site, suggesting that COQ9 might serve to present its bound lipid to COQ7. Collectively, our data define COQ9 as the first, to our knowledge, mammalian TFR structural homolog and suggest that its lipid-binding capacity and association with COQ7 are key features for enabling CoQ biosynthesis.
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http://dx.doi.org/10.1073/pnas.1413128111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4226113PMC
November 2014

Solution structure of a C-terminal fragment (175-257) of CV_0373 protein from Chromobacterium violaceum adopts a winged helix-turn-helix (wHTH) fold.

J Biomol NMR 2014 Nov 1;60(2-3):197-202. Epub 2014 Oct 1.

Department of Chemistry and Biochemistry, and the Northeast Structural Genomics Consortium, Miami University, Oxford, OH, 45056, USA.

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http://dx.doi.org/10.1007/s10858-014-9860-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4928572PMC
November 2014

Solution structure of the free Zα domain of human DLM-1 (ZBP1/DAI), a Z-DNA binding domain.

J Biomol NMR 2014 Nov 31;60(2-3):189-95. Epub 2014 Aug 31.

Department of Chemistry and Biochemistry, and the Northeast Structural Genomics Consortium, Miami University, Oxford, OH, 45056, USA.

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http://dx.doi.org/10.1007/s10858-014-9858-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4527548PMC
November 2014

Solution NMR structures of immunoglobulin-like domains 7 and 12 from obscurin-like protein 1 contribute to the structural coverage of the Human Cancer Protein Interaction Network.

J Struct Funct Genomics 2014 Dec 3;15(4):209-14. Epub 2014 Jul 3.

Department of Chemistry, The State University of New York at Buffalo and Northeast Structural Genomics Consortium, Buffalo, NY, 14260, USA.

High-quality solution NMR structures of immunoglobulin-like domains 7 and 12 from human obscurin-like protein 1 were solved. The two domains share 30% sequence identity and their structures are, as expected, rather similar. The new structures contribute to structural coverage of human cancer associated proteins. Mutations of Arg 812 in domain 7 cause the rare 3-M syndrome, and this site is located in a surface area predicted to be involved in protein-protein interactions.
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http://dx.doi.org/10.1007/s10969-014-9185-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4945113PMC
December 2014

Solution NMR structures of homeodomains from human proteins ALX4, ZHX1, and CASP8AP2 contribute to the structural coverage of the Human Cancer Protein Interaction Network.

J Struct Funct Genomics 2014 Dec 19;15(4):201-7. Epub 2014 Jun 19.

Department of Chemistry, The State University of New York at Buffalo and Northeast Structural Genomics Consortium, Buffalo, NY, 14260, USA.

High-quality solution NMR structures of three homeodomains from human proteins ALX4, ZHX1 and CASP8AP2 were solved. These domains were chosen as targets of a biomedical theme project pursued by the Northeast Structural Genomics Consortium. This project focuses on increasing the structural coverage of human proteins associated with cancer.
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http://dx.doi.org/10.1007/s10969-014-9184-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4239167PMC
December 2014

Polypeptide backbone, C(β) and methyl group resonance assignments of the 24 kDa plectin repeat domain 6 from human protein plectin.

Biomol NMR Assign 2015 Apr 11;9(1):135-138. Epub 2014 Apr 11.

Department of Chemistry, The State University of New York at Buffalo, and Northeast Structural Genomics Consortium, Buffalo, NY 14260, USA.

The 500 kDa protein plectin is essential for the cytoskeletal organization of most mammalian cells and it is up-regulated in some types of cancer. Here, we report nearly complete sequence-specific polypeptide backbone, (13)C(β) and methyl group resonance assignments for 24 kDa human plectin(4403-4606) containing the C-terminal plectin repeat domain 6.
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http://dx.doi.org/10.1007/s12104-014-9559-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4194182PMC
April 2015

Allosteric regulation and substrate activation in cytosolic nucleotidase II from Legionella pneumophila.

FEBS J 2014 Mar 17;281(6):1613-1628. Epub 2014 Feb 17.

Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bangalore 560 064, Karnataka, India.

Unlabelled: Cytosolic nucleotidase II (cN-II) from Legionella pneumophila (Lp) catalyzes the hydrolysis of GMP and dGMP displaying sigmoidal curves, whereas catalysis of IMP hydrolysis displayed a biphasic curve in the initial rate versus substrate concentration plots. Allosteric modulators of mammalian cN-II did not activate LpcN-II although GTP, GDP and the substrate GMP were specific activators. Crystal structures of the tetrameric LpcN-II revealed an activator-binding site at the dimer interface. A double mutation in this allosteric-binding site abolished activation, confirming the structural observations. The substrate GMP acting as an activator, partitioning between the allosteric and active site, is the basis for the sigmoidicity of the initial velocity versus GMP concentration plot. The LpcN-II tetramer showed differences in subunit organization upon activator binding that are absent in the activator-bound human cN-II structure. This is the first observation of a structural change induced by activator binding in cN-II that may be the molecular mechanism for enzyme activation.

Database: The coordinates and structure factors reported in this paper have been submitted to the Protein Data Bank under the accession numbers 2BDE and 4G63. The accession number of GMP complexed LpcN-II is 4OHF.

Structured Digital Abstract: LpcN-II and LpcN-II bind by molecular sieving (View interaction) LpcN-II and LpcN-II bind by x-ray crystallography (View interaction) [Structured digital abstract was added on 5 March 2014 after original online publication].
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http://dx.doi.org/10.1111/febs.12727DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3982195PMC
March 2014

Structural and biochemical characterization of the bilin lyase CpcS from Thermosynechococcus elongatus.

Biochemistry 2013 Dec 19;52(48):8663-76. Epub 2013 Nov 19.

Department of Biological Sciences, University of New Orleans , New Orleans, LA 70148, United States.

Cyanobacterial phycobiliproteins have evolved to capture light energy over most of the visible spectrum due to their bilin chromophores, which are linear tetrapyrroles that have been covalently attached by enzymes called bilin lyases. We report here the crystal structure of a bilin lyase of the CpcS family from Thermosynechococcus elongatus (TeCpcS-III). TeCpcS-III is a 10-stranded β barrel with two alpha helices and belongs to the lipocalin structural family. TeCpcS-III catalyzes both cognate as well as noncognate bilin attachment to a variety of phycobiliprotein subunits. TeCpcS-III ligates phycocyanobilin, phycoerythrobilin, and phytochromobilin to the alpha and beta subunits of allophycocyanin and to the beta subunit of phycocyanin at the Cys82-equivalent position in all cases. The active form of TeCpcS-III is a dimer, which is consistent with the structure observed in the crystal. With the use of the UnaG protein and its association with bilirubin as a guide, a model for the association between the native substrate, phycocyanobilin, and TeCpcS was produced.
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http://dx.doi.org/10.1021/bi401192zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3932240PMC
December 2013

Solution NMR structure of CD1104B from pathogenic Clostridium difficile reveals a distinct α-helical architecture and provides first structural representative of protein domain family PF14203.

J Struct Funct Genomics 2013 Dec 19;14(4):155-60. Epub 2013 Sep 19.

Department of Chemistry, The State University of New York at Buffalo and Northeast Structural Genomics Consortium, Buffalo, NY, 14260, USA.

A high-quality structure of the 68-residue protein CD1104B from Clostridium difficile strain 630 exhibits a distinct all α-helical fold. The structure presented here is the first representative of bacterial protein domain family PF14203 (currently 180 members) of unknown function (DUF4319) and reveals that the side-chains of the only two strictly conserved residues (Glu 8 and Lys 48) form a salt bridge. Moreover, these two residues are located in the vicinity of the largest surface cleft which is predicted to contribute to a surface area involved in protein-protein interactions. This, along with its coding in transposon CTn4, suggests that CD1104B (and very likely all members of Pfam 14203) functions by interacting with other proteins required for the transfer of transposons between different bacterial species.
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http://dx.doi.org/10.1007/s10969-013-9164-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3844015PMC
December 2013

Solution NMR structures provide first structural coverage of the large protein domain family PF08369 and complementary structural coverage of dark operative protochlorophyllide oxidoreductase complexes.

J Struct Funct Genomics 2013 Sep 21;14(3):119-26. Epub 2013 Aug 21.

Department of Chemistry, The State University of New York at Buffalo, Buffalo, NY 14260, USA.

High-quality NMR structures of the C-terminal domain comprising residues 484-537 of the 537-residue protein Bacterial chlorophyll subunit B (BchB) from Chlorobium tepidum and residues 9-61 of 61-residue Asr4154 from Nostoc sp. (strain PCC 7120) exhibit a mixed α/β fold comprised of three α-helices and a small β-sheet packed against second α-helix. These two proteins share 29% sequence similarity and their structures are globally quite similar. The structures of BchB(484-537) and Asr4154(9-61) are the first representative structures for the large protein family (Pfam) PF08369, a family of unknown function currently containing 610 members in bacteria and eukaryotes. Furthermore, BchB(484-537) complements the structural coverage of the dark-operating protochlorophyllide oxidoreductase.
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http://dx.doi.org/10.1007/s10969-013-9159-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3982801PMC
September 2013

Crystal structures of malonyl-coenzyme A decarboxylase provide insights into its catalytic mechanism and disease-causing mutations.

Structure 2013 Jul 20;21(7):1182-92. Epub 2013 Jun 20.

Structural Genomics Consortium, University of Oxford, Oxford OX3 7DQ, UK.

Malonyl-coenzyme A decarboxylase (MCD) is found from bacteria to humans, has important roles in regulating fatty acid metabolism and food intake, and is an attractive target for drug discovery. We report here four crystal structures of MCD from human, Rhodopseudomonas palustris, Agrobacterium vitis, and Cupriavidus metallidurans at up to 2.3 Å resolution. The MCD monomer contains an N-terminal helical domain involved in oligomerization and a C-terminal catalytic domain. The four structures exhibit substantial differences in the organization of the helical domains and, consequently, the oligomeric states and intersubunit interfaces. Unexpectedly, the MCD catalytic domain is structurally homologous to those of the GCN5-related N-acetyltransferase superfamily, especially the curacin A polyketide synthase catalytic module, with a conserved His-Ser/Thr dyad important for catalysis. Our structures, along with mutagenesis and kinetic studies, provide a molecular basis for understanding pathogenic mutations and catalysis, as well as a template for structure-based drug design.
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http://dx.doi.org/10.1016/j.str.2013.05.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3701320PMC
July 2013
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