Eric Hampton

The nuclear receptor LXR is a glucose sensor

The liver has a central role in glucose homeostasis, as it has the distinctive ability to produce and consume glucose. On feeding, glucose influx triggers gene expression changes in hepatocytes to suppress endogenous glucose production and convert excess glucose into glycogen or fatty acids to be stored in adipose tissue. This process is controlled by insulin, although debate exists as to whether insulin acts directly or indirectly on the liver. In addition to stimulating pancreatic insulin release, glucose also regulates the activity of ChREBP, a transcription factor that modulates lipogenesis. Here we describe another mechanism whereby glucose determines its own fate: we show that glucose binds and stimulates the transcriptional activity of the liver X receptor (LXR), a nuclear receptor that coordinates hepatic lipid metabolism. d-Glucose and d-glucose-6-phosphate are direct agonists of both LXR-α and LXR-β. Glucose activates LXR at physiological concentrations expected in the liver and induces expression of LXR target genes with efficacy similar to that of oxysterols, the known LXR ligands. Cholesterol homeostasis genes that require LXR for expression are upregulated in liver and intestine of fasted mice re-fed with a glucose diet, indicating that glucose is an endogenous LXR ligand. Our results identify LXR as a transcriptional switch that integrates hepatic glucose metabolism and fatty acid synthesis.

Secreted PCSK9 promotes LDL receptor degradation independently of proteolytic activity

PCSK9 (proprotein convertase subtilisin/kexin 9) is a secreted serine protease that regulates cholesterol homoeostasis by inducing post-translational degradation of hepatic LDL-R [LDL (low-density lipoprotein) receptor]. Intramolecular autocatalytic processing of the PCSK9 zymogen in the endoplasmic reticulum results in a tightly associated complex between the prodomain and the catalytic domain. Although the autocatalytic processing event is required for proper secretion of PCSK9, the requirement of proteolytic activity in the regulation of LDL-R is currently unknown. Co-expression of the prodomain and the catalytic domain in trans allowed for production of a catalytically inactive secreted form of PCSK9. This catalytically inactive PCSK9 was characterized and shown to be functionally equivalent to the wild-type protein in lowering cellular LDL uptake and LDL-R levels. These findings suggest that, apart from autocatalytic processing, the protease activity of PCSK9 is not necessary for LDL-R regulation.

The self-inhibited structure of full-length PCSK9 at 1.9 Å reveals structural homology with resistin within the C-terminal domain

Mutations in proprotein convertase subtilisin/kexin type 9 (PCSK9) are strongly associated with levels of low-density lipoprotein cholesterol in the blood plasma and, thereby, occurrence or resistance to atherosclerosis and coronary heart disease. Despite this importance, relatively little is known about the biology of PCSK9. Here, the crystal structure of a full-length construct of PCSK9 solved to 1.9-Å resolution is presented. The structure contains a fully folded C-terminal cysteine-rich domain (CRD), showing a distinct structural similarity to the resistin homotrimer, a small cytokine associated with obesity and diabetes. This structural relationship between the CRD of PCSK9 and the resistin family is not observed in primary sequence comparisons and strongly suggests a distant evolutionary link between the two molecules. This three-dimensional homology provides insight into the function of PCSK9 at the molecular level and will help to dissect the link between PCSK9 and CHD.

Switch-mediated activation and retargeting of CAR-T cells for B-cell malignancies.

Significance Chimeric antigen receptor T (CAR-T) cell therapy has produced promising results in clinical trials but has been challenged by the inability to control engineered cells once infused into the patient. Here we present a generalizable method of controlling CAR-T cells using peptide-engrafted antibody-based molecular switches that act as a bridge between the target cell and CAR-T cell. We show that switches specific for CD19 govern the activity, tissue-homing, cytokine release, and phenotype of switchable CAR-T cells in a dose-titratable manner using xenograft mouse models of B-cell leukemia. We expect that this method of tuning CAR-T cell responses will provide improved safety and versatility of CAR–T-cell therapy in the clinic. Chimeric antigen receptor T (CAR-T) cell therapy has produced impressive results in clinical trials for B-cell malignancies. However, safety concerns related to the inability to control CAR-T cells once infused into the patient remain a significant challenge. Here we report the engineering of recombinant antibody-based bifunctional switches that consist of a tumor antigen-specific Fab molecule engrafted with a peptide neo-epitope, which is bound exclusively by a peptide-specific switchable CAR-T cell (sCAR-T). The switch redirects the activity of the bio-orthogonal sCAR-T cells through the selective formation of immunological synapses, in which the sCAR-T cell, switch, and target cell interact in a structurally defined and temporally controlled manner. Optimized switches specific for CD19 controlled the activity, tissue-homing, cytokine release, and phenotype of sCAR-T cells in a dose-titratable manner in a Nalm-6 xenograft rodent model of B-cell leukemia. The sCAR–T-cell dosing regimen could be tuned to provide efficacy comparable to the corresponding conventional CART-19, but with lower cytokine levels, thereby offering a method of mitigating cytokine release syndrome in clinical translation. Furthermore, we demonstrate that this methodology is readily adaptable to targeting CD20 on cancer cells using the same sCAR-T cell, suggesting that this approach may be broadly applicable to heterogeneous and resistant tumor populations, as well as other liquid and solid tumor antigens.

Crystal structures of two novel dye-decolorizing peroxidases reveal a beta-barrel fold with a conserved heme-binding motif.

BtDyP from Bacteroides thetaiotaomicron (strain VPI‐5482) and TyrA from Shewanella oneidensis are dye‐decolorizing peroxidases (DyPs), members of a new family of heme‐dependent peroxidases recently identified in fungi and bacteria. Here, we report the crystal structures of BtDyP and TyrA at 1.6 and 2.7 Å, respectively. BtDyP assembles into a hexamer, while TyrA assembles into a dimer; the dimerization interface is conserved between the two proteins. Each monomer exhibits a two‐domain, α+β ferredoxin‐like fold. A site for heme binding was identified computationally, and modeling of a heme into the proposed active site allowed for identification of residues likely to be functionally important. Structural and sequence comparisons with other DyPs demonstrate a conservation of putative heme‐binding residues, including an absolutely conserved histidine. Isothermal titration calorimetry experiments confirm heme binding, but with a stoichiometry of 0.3:1 (heme:protein). Proteins 2007.

Crystal structure of the global regulatory protein CsrA from Pseudomonas putida at 2.05 Å resolution reveals a new fold

Chris Rife, Robert Schwarzenbacher, Daniel McMullan, Polat Abdubek, Eileen Ambing, Herbert Axelrod, Tanya Biorac, Jaume M. Canaves, Hsiu-Ju Chiu, Ashley M. Deacon, Michael DiDonato, Marc-André Elsliger, Adam Godzik, Carina Grittini, Slawomir K. Grzechnik, Joanna Hale, Eric Hampton, Gye Won Han, Justin Haugen, Michael Hornsby, Lukasz Jaroszewski, Heath E. Klock, Eric Koesema, Andreas Kreusch, Peter Kuhn, Scott A. Lesley, Mitchell D. Miller, Kin Moy, Edward Nigoghossian, Jessica Paulsen, Kevin Quijano, Ron Reyes, Eric Sims, Glen Spraggon, Raymond C. Stevens, Henry van den Bedem, Jeff Velasquez, Juli Vincent, Aprilfawn White, Guenter Wolf, Qingping Xu, Keith O. Hodgson, John Wooley, and Ian A. Wilson* The Joint Center for Structural Genomics Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, California The University of California, San Diego, La Jolla, California The Genomics Institute of the Novartis Research Foundation, San Diego, California The Scripps Research Institute, La Jolla, California

Crystal structure of a tandem cystathionine-β-synthase (CBS) domain protein (TM0935) from Thermotoga maritima at 1.87 Å resolution

Mitchell D. Miller, Robert Schwarzenbacher, Frank von Delft, Polat Abdubek, Eileen Ambing, Tanya Biorac, Linda S. Brinen, Jaume M. Canaves, Jamison Cambell, Hsiu-Ju Chiu, Xiaoping Dai, Ashley M. Deacon, Mike DiDonato, Marc-André Elsliger, Said Eshagi, Ross Floyd, Adam Godzik, Carina Grittini, Slawomir K. Grzechnik, Eric Hampton, Lukasz Jaroszewski, Cathy Karlak, Heath E. Klock, Eric Koesema, John S. Kovarik, Andreas Kreusch, Peter Kuhn, Scott A. Lesley, Inna Levin, Daniel McMullan, Timothy M. McPhillips, Andrew Morse, Kin Moy, Jie Ouyang, Rebecca Page, Kevin Quijano, Alyssa Robb, Glen Spraggon, Raymond C. Stevens, Henry van den Bedem, Jeff Velasquez, Juli Vincent, Xianhong Wang, Bill West, Guenter Wolf, Qingping Xu, Keith O. Hodgson, John Wooley, and Ian A. Wilson* Joint Center for Structural Genomics, Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park California Genomics Institute of the Novartis Research Foundation, San Diego, California San Diego Supercomputer Center, La Jolla, California University of California, San Diego, La Jolla, California Scripps Research Institute, La Jolla, California

Crystal structure of a PIN (PilT N‐terminus) domain (AF0591) from Archaeoglobus fulgidus at 1.90 Å resolution

Inna Levin, Robert Schwarzenbacher, Rebecca Page, Polat Abdubek, Eileen Ambing, Tanya Biorac, Linda S. Brinen, Jamison Campbell, Jaume M. Canaves, Hsiu-Ju Chiu, Xiaoping Dai, Ashley M. Deacon, Mike DiDonato, Marc-André Elsliger, Ross Floyd, Adam Godzik, Carina Grittini, Slawomir K. Grzechnik, Eric Hampton, Lukasz Jaroszewski, Cathy Karlak, Heath E. Klock, Eric Koesema, John S. Kovarik, Andreas Kreusch, Peter Kuhn, Scott A. Lesley, Daniel McMullan, Timothy M. McPhillips, Mitchell D. Miller, Andrew Morse, Kin Moy, Jie Ouyang, Kevin Quijano, Ron Reyes, Fred Rezezadeh, Alyssa Robb, Eric Sims, Glen Spraggon, Raymond C. Stevens, Henry van den Bedem, Jeff Velasquez, Juli Vincent, Frank von Delft, Xianhong Wang, Bill West, Guenter Wolf, Qingping Xu, Keith O. Hodgson, John Wooley, and Ian A. Wilson* Joint Center for Structural Genomics, Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park California Genomics Institute of the Novartis Research Foundation, San Diego, California San Diego Supercomputer Center, La Jolla, California University of California, San Diego, La Jolla, California Scripps Research Institute, La Jolla, California

Crystal structure of acireductone dioxygenase (ARD) from Mus musculus at 2.06 Å resolution

Qingping Xu, Robert Schwarzenbacher, S. Sri Krishna, Daniel McMullan, Sanjay Agarwalla, Kevin Quijano, Polat Abdubek, Eileen Ambing, Herbert Axelrod, Tanya Biorac, Jaume M. Canaves, Hsiu-Ju Chiu, Marc-André Elsliger, Carina Grittini, Slawomir K. Grzechnik, Michael DiDonato, Joanna Hale, Eric Hampton, Gye Won Han, Justin Haugen, MichaelHornsby, Lukasz Jaroszewski, Heath E. Klock, Mark W. Knuth, Eric Koesema, Andreas Kreusch, Peter Kuhn, Mitchell D. Miller, Kin Moy, Edward Nigoghossian, Jessica Paulsen, Ron Reyes, Chris Rife, Glen Spraggon, Raymond C. Stevens, Henry van den Bedem, Jeff Velasquez, Aprilfawn White, Guenter Wolf, Keith O. Hodgson, John Wooley, Ashley M. Deacon, Adam Godzik, Scott A. Lesley, and Ian A. Wilson* The Joint Center for Structural Genomics Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, California The University of California San Diego, La Jolla, California The Genomics Institute of the Novartis Research Foundation, San Diego, California The Scripps Research Institute, La Jolla, California

Crystal structure of a novel manganese-containing cupin (TM1459) from Thermotoga maritima at 1.65 Å resolution

Lukasz Jaroszewski, Robert Schwarzenbacher, Frank von Delft, Daniel McMullan, Linda S. Brinen, Jaume M. Canaves, Xiaoping Dai, Ashley M. Deacon, Mike DiDonato, Marc-André Elsliger, Said Eshagi, Ross Floyd, Adam Godzik, Carina Grittini, Slawomir K. Grzechnik, Eric Hampton, Inna Levin, Cathy Karlak, Heath E. Klock, Eric Koesema, John S. Kovarik, Andreas Kreusch, Peter Kuhn, Scott A. Lesley, Timothy M. McPhillips, Mitchell D. Miller, Andrew Morse, Kin Moy, Jie Ouyang, Rebecca Page, Kevin Quijano, Ron Reyes, Fred Rezezadeh, Alyssa Robb, Eric Sims, Glen Spraggon, Raymond C. Stevens, Henry van den Bedem, Jeff Velasquez, Juli Vincent, Xianhong Wang, Bill West, Guenter Wolf, Qingping Xu, Keith O. Hodgson, John Wooley, and Ian A. Wilson* The Joint Center for Structural Genomics, Stanford Synchrotron Radiation Laboratory, Stanford University, 2575 Sand Hill Rd, Menlo Park, California The Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Dr., San Diego, California The San Diego Supercomputer Center, 9500 Gilman Drive, La Jolla, California The University of California, San Diego, 9500 Gilman Drive, La Jolla, California The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, California