Publications by authors named "Stuart A Sievers"

19 Publications

  • Page 1 of 1

Activation of CAR and non-CAR T cells within the tumor microenvironment following CAR T cell therapy.

JCI Insight 2020 06 18;5(12). Epub 2020 Jun 18.

Center for Immuno-Oncology and.

Mechanisms of chimeric antigen receptor (CAR) T cell-mediated antitumor immunity and toxicity remain poorly characterized because few studies examine the intact tumor microenvironment (TME) following CAR T cell infusion. Axicabtagene ciloleucel is an autologous anti-CD19 CAR T cell therapy approved for patients with large B cell lymphoma. We devised multiplex immunostaining and ISH assays to interrogate CAR T cells and other immune cell infiltrates in biopsies of diffuse large B cell lymphoma following axicabtagene ciloleucel infusion. We found that a majority of intratumoral CAR T cells expressed markers of T cell activation but, unexpectedly, constituted ≤5% of all T cells within the TME 5 days or more after therapy. Large numbers of T cells without CAR were also activated within the TME after axicabtagene ciloleucel infusion; these cells were positive for Ki-67, IFN-γ, granzyme B (GzmB), and/or PD-1 and were found at the highest levels in biopsies with CAR T cells. Additionally, non-CAR immune cells were the exclusive source of IL-6, a cytokine associated with cytokine release syndrome, and were found at their highest numbers in biopsies with CAR T cells. These data suggest that intratumoral CAR T cells are associated with non-CAR immune cell activation within the TME with both beneficial and pathological effects.
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http://dx.doi.org/10.1172/jci.insight.134612DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7406247PMC
June 2020

Crystal structures of amyloidogenic segments of human transthyretin.

Protein Sci 2018 07 13;27(7):1295-1303. Epub 2018 Jun 13.

Departments of Biological Chemistry and Chemistry and Biochemistry, Molecular Biology Institute, Box 951570, UCLA, Howard Hughes Medical Institute, UCLA-DOE Institute, Los Angeles, California, 90095-1570.

Amyloid diseases are characterized by the deposition of proteins in the form of amyloid fibrils, in organs that eventually fail. The development of effective drug candidates follows from the understanding of the molecular processes that lead to protein aggregation. Here, we study amyloidogenic segments of transthyretin (TTR). TTR is a transporter of thyroxine and retinol in the blood and cerebrospinal fluid. When mutated and/or as a result of aging, TTR aggregates into amyloid fibrils that accumulate in organs such as the heart. Recently, we reported two amyloidogenic segments that drive amyloid aggregation. Here, we report the crystal structure of another six amyloidogenic segments of TTR. We found that the segments from the C-terminal region of TTR form in-register steric-zippers with highly-interdigitated, wet interfaces, whereas the β-strand B from the N-terminal region of TTR forms an out-of-register assembly, previously associated with oligomeric formation. Our results contribute fundamental information for understanding the mechanism of aggregation of TTR.
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http://dx.doi.org/10.1002/pro.3420DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6032358PMC
July 2018

Function of Novel Anti-CD19 Chimeric Antigen Receptors with Human Variable Regions Is Affected by Hinge and Transmembrane Domains.

Mol Ther 2017 11 27;25(11):2452-2465. Epub 2017 Jul 27.

Experimental Transplantation and Immunology Branch, National Cancer Institute, Bethesda, MD 20892, USA. Electronic address:

Anti-CD19 chimeric antigen receptor (CAR) T cells have caused remissions of B cell malignancies, but problems including cytokine-mediated toxicity and short persistence of CAR T cells in vivo might limit the effectiveness of anti-CD19 CAR T cells. Anti-CD19 CARs that have been tested clinically had single-chain variable fragments (scFvs) derived from murine antibodies. We have designed and constructed novel anti-CD19 CARs containing a scFv with fully human variable regions. T cells expressing these CARs specifically recognized CD19 target cells and carried out functions including degranulation, cytokine release, and proliferation. We compared CARs with CD28 costimulatory moieties along with hinge and transmembrane domains from either the human CD28 molecule or the human CD8α molecule. Compared with T cells expressing CARs with CD28 hinge and transmembrane domains, T cells expressing CARs with CD8α hinge and transmembrane domains produced lower levels of cytokines and exhibited lower levels of activation-induced cell death (AICD). Importantly, CARs with hinge and transmembrane regions from either CD8α or CD28 had similar abilities to eliminate established tumors in mice. In anti-CD19 CARs with CD28 costimulatory moieties, lower levels of inflammatory cytokine production and AICD are potential clinical advantages of CD8α hinge and transmembrane domains over CD28 hinge and transmembrane domains.
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http://dx.doi.org/10.1016/j.ymthe.2017.07.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5675490PMC
November 2017

Coexistence of potent HIV-1 broadly neutralizing antibodies and antibody-sensitive viruses in a viremic controller.

Sci Transl Med 2017 01;9(373)

Laboratory of Molecular Immunology, Rockefeller University, New York, NY 10065, USA.

Some HIV-1-infected patients develop broad and potent HIV-1 neutralizing antibodies (bNAbs) that when passively transferred to mice or macaques can treat or prevent infection. However, bNAbs typically fail to neutralize coexisting autologous viruses due to antibody-mediated selection against sensitive viral strains. We describe an HIV-1 controller expressing HLA-B57*01 and HLA-B27*05 who maintained low viral loads for 30 years after infection and developed broad and potent serologic activity against HIV-1. Neutralization was attributed to three different bNAbs targeting nonoverlapping sites on the HIV-1 envelope trimer (Env). One of the three, BG18, an antibody directed against the glycan-V3 portion of Env, is the most potent member of this class reported to date and, as revealed by crystallography and electron microscopy, recognizes HIV-1 Env in a manner that is distinct from other bNAbs in this class. Single-genome sequencing of HIV-1 from serum samples obtained over a period of 9 years showed a diverse group of circulating viruses, 88.5% (31 of 35) of which remained sensitive to at least one of the temporally coincident autologous bNAbs and the individual's serum. Thus, bNAb-sensitive strains of HIV-1 coexist with potent neutralizing antibodies that target the virus and may contribute to control in this individual. When administered as a mix, the three bNAbs controlled viremia in HIV-1-infected humanized mice. Our finding suggests that combinations of bNAbs may contribute to control of HIV-1 infection.
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http://dx.doi.org/10.1126/scitranslmed.aal2144DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5467220PMC
January 2017

Structural basis for germline antibody recognition of HIV-1 immunogens.

Elife 2016 Mar 21;5. Epub 2016 Mar 21.

Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, United States.

Efforts to elicit broadly neutralizing antibodies (bNAbs) against HIV-1 require understanding germline bNAb recognition of HIV-1 envelope glycoprotein (Env). The VRC01-class bNAb family derived from the VH1-2*02 germline allele arose in multiple HIV-1-infected donors, yet targets the CD4-binding site on Env with common interactions. Modified forms of the 426c Env that activate germline-reverted B cell receptors are candidate immunogens for eliciting VRC01-class bNAbs. We present structures of germline-reverted VRC01-class bNAbs alone and complexed with 426c-based gp120 immunogens. Germline bNAb-426c gp120 complexes showed preservation of VRC01-class signature residues and gp120 contacts, but detectably different binding modes compared to mature bNAb-gp120 complexes. Unlike typical antibody-antigen interactions, VRC01-class germline antibodies exhibited preformed antigen-binding conformations for recognizing immunogens. Affinity maturation introduced substitutions increasing induced-fit recognition and electropositivity, potentially to accommodate negatively-charged complex-type N-glycans on gp120. These results provide general principles relevant to the unusual evolution of VRC01-class bNAbs and guidelines for structure-based immunogen design.
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http://dx.doi.org/10.7554/eLife.13783DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4811768PMC
March 2016

A New Glycan-Dependent CD4-Binding Site Neutralizing Antibody Exerts Pressure on HIV-1 In Vivo.

PLoS Pathog 2015 Oct 30;11(10):e1005238. Epub 2015 Oct 30.

Laboratory of Molecular Immunology, The Rockefeller University, New York, New York, United States of America; Howard Hughes Medical Institute, Chevy Chase, Maryland, United States of America.

The CD4 binding site (CD4bs) on the envelope glycoprotein is a major site of vulnerability that is conserved among different HIV-1 isolates. Many broadly neutralizing antibodies (bNAbs) to the CD4bs belong to the VRC01 class, sharing highly restricted origins, recognition mechanisms and viral escape pathways. We sought to isolate new anti-CD4bs bNAbs with different origins and mechanisms of action. Using a gp120 2CC core as bait, we isolated antibodies encoded by IGVH3-21 and IGVL3-1 genes with long CDRH3s that depend on the presence of the N-linked glycan at position-276 for activity. This binding mode is similar to the previously identified antibody HJ16, however the new antibodies identified herein are more potent and broad. The most potent variant, 179NC75, had a geometric mean IC80 value of 0.42 μg/ml against 120 Tier-2 HIV-1 pseudoviruses in the TZM.bl assay. Although this group of CD4bs glycan-dependent antibodies can be broadly and potently neutralizing in vitro, their in vivo activity has not been tested to date. Here, we report that 179NC75 is highly active when administered to HIV-1-infected humanized mice, where it selects for escape variants that lack a glycan site at position-276. The same glycan was absent from the virus isolated from the 179NC75 donor, implying that the antibody also exerts selection pressure in humans.
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http://dx.doi.org/10.1371/journal.ppat.1005238DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4627763PMC
October 2015

Antibody engineering for increased potency, breadth and half-life.

Curr Opin HIV AIDS 2015 May;10(3):151-9

aDivision of Biology and Biological Engineering, California Institute of Technology, 1200 East California Boulevard, Pasadena, CA 91125, USA bHoward Hughes Medical Institute, Chevy Chase, MD 20815-6789, USA.

Purpose Of Review: This review highlights recent developments in HIV-1 antibody engineering and discusses the effects of increased polyreactivity on serum half-lives of engineered antibodies.

Recent Findings: Recent studies have uncovered a wealth of information about the relationship between the sequences and efficacies of anti-HIV-1 antibodies through a combination of bioinformatics, structural characterization and in vivo studies. This knowledge has stimulated efforts to enhance antibody breadth and potency for therapeutic use. Although some engineered antibodies have shown increased polyreactivity and short half-lives, promising efforts are circumventing these problems.

Summary: Antibodies are desirable as therapeutics due to their ability to recognize targets with both specificity and high affinity. Furthermore, the ability of antibodies to stimulate Fc-mediated effector functions can increase their utility. Thus, mAbs have become central to strategies for the treatment of various diseases. Using both targeted and library-based approaches, antibodies can be engineered to improve their therapeutic properties. This article will discuss recent antibody engineering efforts to improve the breadth and potency of anti-HIV-1 antibodies. The polyreactivity of engineered HIV-1 bNAbs and the effect on serum half-life will be explored along with strategies to overcome problems introduced by engineering antibodies. Finally, advances in creating bispecific anti-HIV-1 reagents are discussed.
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http://dx.doi.org/10.1097/COH.0000000000000148DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4465343PMC
May 2015

Structure-based design of functional amyloid materials.

J Am Chem Soc 2014 Dec 19;136(52):18044-51. Epub 2014 Dec 19.

UCLA-DOE Institute, and Department of Chemistry and Biochemistry, University of California , Los Angeles, California 90095, United States.

Amyloid fibers, once exclusively associated with disease, are acquiring utility as a class of biological nanomaterials. Here we introduce a method that utilizes the atomic structures of amyloid peptides, to design materials with versatile applications. As a model application, we designed amyloid fibers capable of capturing carbon dioxide from flue gas, to address the global problem of excess anthropogenic carbon dioxide. By measuring dynamic separation of carbon dioxide from nitrogen, we show that fibers with designed amino acid sequences double the carbon dioxide binding capacity of the previously reported fiber formed by VQIVYK from Tau protein. In a second application, we designed fibers that facilitate retroviral gene transfer. By measuring lentiviral transduction, we show that designed fibers exceed the efficiency of polybrene, a commonly used enhancer of transduction. The same procedures can be adapted to the design of countless other amyloid materials with a variety of properties and uses.
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http://dx.doi.org/10.1021/ja509648uDOI Listing
December 2014

Aggregation-triggering segments of SOD1 fibril formation support a common pathway for familial and sporadic ALS.

Proc Natl Acad Sci U S A 2014 Jan 16;111(1):197-201. Epub 2013 Dec 16.

Howard Hughes Medical Institute, UCLA-DOE Institute for Genomics and Proteomics, and Department of Biological Chemistry, University of California, Los Angeles, CA 90095-1570.

ALS is a terminal disease of motor neurons that is characterized by accumulation of proteinaceous deposits in affected cells. Pathological deposition of mutated Cu/Zn superoxide dismutase (SOD1) accounts for ∼20% of the familial ALS (fALS) cases. However, understanding the molecular link between mutation and disease has been difficult, given that more than 140 different SOD1 mutants have been observed in fALS patients. In addition, the molecular origin of sporadic ALS (sALS) is unclear. By dissecting the amino acid sequence of SOD1, we identified four short segments with a high propensity for amyloid fibril formation. We find that fALS mutations in these segments do not reduce their propensity to form fibrils. The atomic structures of two fibril-forming segments from the C terminus, (101)DSVISLS(107) and (147)GVIGIAQ(153), reveal tightly packed β-sheets with steric zipper interfaces characteristic of the amyloid state. Based on these structures, we conclude that both C-terminal segments are likely to form aggregates if available for interaction. Proline substitutions in (101)DSVISLS(107) and (147)GVIGIAQ(153) impaired nucleation and fibril growth of full-length protein, confirming that these segments participate in aggregate formation. Our hypothesis is that improper protein maturation and incompletely folded states that render these aggregation-prone segments available for interaction offer a common molecular pathway for sALS and fALS.
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http://dx.doi.org/10.1073/pnas.1320786110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3890817PMC
January 2014

Towards a pharmacophore for amyloid.

PLoS Biol 2011 Jun 14;9(6):e1001080. Epub 2011 Jun 14.

Howard Hughes Medical Institute, UCLA-DOE Institute for Genomics and Proteomics, Department of Biological Chemistry, University of California, Los Angeles, California, United States of America.

Diagnosing and treating Alzheimer's and other diseases associated with amyloid fibers remains a great challenge despite intensive research. To aid in this effort, we present atomic structures of fiber-forming segments of proteins involved in Alzheimer's disease in complex with small molecule binders, determined by X-ray microcrystallography. The fiber-like complexes consist of pairs of β-sheets, with small molecules binding between the sheets, roughly parallel to the fiber axis. The structures suggest that apolar molecules drift along the fiber, consistent with the observation of nonspecific binding to a variety of amyloid proteins. In contrast, negatively charged orange-G binds specifically to lysine side chains of adjacent sheets. These structures provide molecular frameworks for the design of diagnostics and drugs for protein aggregation diseases.
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http://dx.doi.org/10.1371/journal.pbio.1001080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3114762PMC
June 2011

Structure-based design of non-natural amino-acid inhibitors of amyloid fibril formation.

Nature 2011 Jun 15;475(7354):96-100. Epub 2011 Jun 15.

Department of Biological Chemistry, Howard Hughes Medical Institute, UCLA, Box 951970, Los Angeles, California 90095-1570, USA.

Many globular and natively disordered proteins can convert into amyloid fibrils. These fibrils are associated with numerous pathologies as well as with normal cellular functions, and frequently form during protein denaturation. Inhibitors of pathological amyloid fibril formation could be useful in the development of therapeutics, provided that the inhibitors were specific enough to avoid interfering with normal processes. Here we show that computer-aided, structure-based design can yield highly specific peptide inhibitors of amyloid formation. Using known atomic structures of segments of amyloid fibrils as templates, we have designed and characterized an all-D-amino-acid inhibitor of the fibril formation of the tau protein associated with Alzheimer's disease, and a non-natural L-amino-acid inhibitor of an amyloid fibril that enhances sexual transmission of human immunodeficiency virus. Our results indicate that peptides from structure-based designs can disrupt the fibril formation of full-length proteins, including those, such as tau protein, that lack fully ordered native structures. Because the inhibiting peptides have been designed on structures of dual-β-sheet 'steric zippers', the successful inhibition of amyloid fibril formation strengthens the hypothesis that amyloid spines contain steric zippers.
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http://dx.doi.org/10.1038/nature10154DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4073670PMC
June 2011

Molecular basis for insulin fibril assembly.

Proc Natl Acad Sci U S A 2009 Nov 28;106(45):18990-5. Epub 2009 Oct 28.

Howard Hughes Medical Institute, UCLA-DOE Institute for Genomics and Proteomics, Los Angeles CA 90095-1570, USA.

In the rare medical condition termed injection amyloidosis, extracellular fibrils of insulin are observed. We found that the segment of the insulin B-chain with sequence LVEALYL is the smallest segment that both nucleates and inhibits the fibrillation of full-length insulin in a molar ratio-dependent manner, suggesting that this segment is central to the cross-beta spine of the insulin fibril. In isolation from the rest of the protein, LVEALYL forms microcrystalline aggregates with fibrillar morphology, the structure of which we determined to 1 A resolution. The LVEALYL segments are stacked into pairs of tightly interdigitated beta-sheets, each pair displaying the dry steric zipper interface typical of amyloid-like fibrils. This structure leads to a model for fibrils of human insulin consistent with electron microscopic, x-ray fiber diffraction, and biochemical studies.
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http://dx.doi.org/10.1073/pnas.0910080106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2776439PMC
November 2009

Atomic structures of IAPP (amylin) fusions suggest a mechanism for fibrillation and the role of insulin in the process.

Protein Sci 2009 Jul;18(7):1521-30

Howard Hughes Medical Institute, UCLA-DOE Institute of Genomics and Proteomics, Los Angeles, California 90095-1570, USA.

Islet Amyloid Polypeptide (IAPP or amylin) is a peptide hormone produced and stored in the beta-islet cells of the pancreas along with insulin. IAPP readily forms amyloid fibrils in vitro, and the deposition of fibrillar IAPP has been correlated with the pathology of type II diabetes. The mechanism of the conversion that IAPP undergoes from soluble to fibrillar forms has been unclear. By chaperoning IAPP through fusion to maltose binding protein, we find that IAPP can adopt a alpha-helical structure at residues 8-18 and 22-27 and that molecules of IAPP dimerize. Mutational analysis suggests that this dimerization is on the pathway to fibrillation. The structure suggests how IAPP may heterodimerize with insulin, which we confirmed by protein crosslinking. Taken together, these experiments suggest the helical dimerization of IAPP accelerates fibril formation and that insulin impedes fibrillation by blocking the IAPP dimerization interface.
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http://dx.doi.org/10.1002/pro.145DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2775219PMC
July 2009

Atomic structure of the cross-beta spine of islet amyloid polypeptide (amylin).

Protein Sci 2008 Sep 12;17(9):1467-74. Epub 2008 Jun 12.

Howard Hughes Medical Institute, UCLA-DOE Institute for Genomics and Proteomics, Los Angeles, California 90095-1570, USA.

Human islet amyloid polypeptide (IAPP or amylin) is a 37-residue hormone found as fibrillar deposits in pancreatic extracts of nearly all type II diabetics. Although the cellular toxicity of IAPP has been established, the structure of the fibrillar form found in these deposits is unknown. Here we have crystallized two segments from IAPP, which themselves form amyloid-like fibrils. The atomic structures of these two segments, NNFGAIL and SSTNVG, were determined, and form the basis of a model for the most commonly observed, full-length IAPP polymorph.
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http://dx.doi.org/10.1110/ps.036509.108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2525530PMC
September 2008

Draft crystal structure of the vault shell at 9-A resolution.

PLoS Biol 2007 Nov;5(11):e318

Howard Hughes Medical Institute, University of California Los Angeles, Los Angeles, California, United States of America.

Vaults are the largest known cytoplasmic ribonucleoprotein structures and may function in innate immunity. The vault shell self-assembles from 96 copies of major vault protein and encapsulates two other proteins and a small RNA. We crystallized rat liver vaults and several recombinant vaults, all among the largest non-icosahedral particles to have been crystallized. The best crystals thus far were formed from empty vaults built from a cysteine-tag construct of major vault protein (termed cpMVP vaults), diffracting to about 9-A resolution. The asymmetric unit contains a half vault of molecular mass 4.65 MDa. X-ray phasing was initiated by molecular replacement, using density from cryo-electron microscopy (cryo-EM). Phases were improved by density modification, including concentric 24- and 48-fold rotational symmetry averaging. From this, the continuous cryo-EM electron density separated into domain-like blocks. A draft atomic model of cpMVP was fit to this improved density from 15 domain models. Three domains were adapted from a nuclear magnetic resonance substructure. Nine domain models originated in ab initio tertiary structure prediction. Three C-terminal domains were built by fitting poly-alanine to the electron density. Locations of loops in this model provide sites to test vault functions and to exploit vaults as nanocapsules.
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http://dx.doi.org/10.1371/journal.pbio.0050318DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2229873PMC
November 2007

Atomic structures of amyloid cross-beta spines reveal varied steric zippers.

Nature 2007 May 29;447(7143):453-7. Epub 2007 Apr 29.

Howard Hughes Medical Institute, UCLA-DOE Institute of Genomics and Proteomics, Los Angeles, California 90095-1570, USA.

Amyloid fibrils formed from different proteins, each associated with a particular disease, contain a common cross-beta spine. The atomic architecture of a spine, from the fibril-forming segment GNNQQNY of the yeast prion protein Sup35, was recently revealed by X-ray microcrystallography. It is a pair of beta-sheets, with the facing side chains of the two sheets interdigitated in a dry 'steric zipper'. Here we report some 30 other segments from fibril-forming proteins that form amyloid-like fibrils, microcrystals, or usually both. These include segments from the Alzheimer's amyloid-beta and tau proteins, the PrP prion protein, insulin, islet amyloid polypeptide (IAPP), lysozyme, myoglobin, alpha-synuclein and beta(2)-microglobulin, suggesting that common structural features are shared by amyloid diseases at the molecular level. Structures of 13 of these microcrystals all reveal steric zippers, but with variations that expand the range of atomic architectures for amyloid-like fibrils and offer an atomic-level hypothesis for the basis of prion strains.
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http://dx.doi.org/10.1038/nature05695DOI Listing
May 2007

Identification and biochemical characterization of serine hydroxymethyl transferase in the hydrogenosome of Trichomonas vaginalis.

Eukaryot Cell 2006 Dec 15;5(12):2072-8. Epub 2006 Sep 15.

Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, 609 Charles E. Young Drive East, Los Angeles, CA 90095-1489, USA.

Serine hydroxymethyl transferase (SHMT) is a pyridoxal phosphate (PLP)-dependent enzyme that catalyzes the reversible conversion of serine and tetrahydrofolate to glycine and methylenetetrahydrofolate. We have identified a single gene encoding SHMT in the genome of Trichomonas vaginalis, an amitochondriate, deep-branching unicellular protist. The protein possesses a putative N-terminal hydrogenosomal presequence and was shown to localize to hydrogensomes by immunofluorescence analysis, providing evidence of amino acid metabolism in this unusual organelle. In contrast to the tetrameric SHMT that exists in the mammalian host, we found that the T. vaginalis SHMT is a homodimer, as found in prokaryotes. All examined SHMT contain an 8-amino-acid conserved sequence, VTTTTHKT, containing the active-site lysyl residue (Lys 251 in TvSHMT) that forms an internal aldimine with PLP. We mutated this Lys residue to Arg and Gln and examined structural and catalytic properties of the wild-type and mutant enzymes in comparison to that reported for the mammalian protein. The oligomeric structure of the mutant K251R and K251Q TvSHMT was not affected, in contrast to that observed for comparable mutations in the mammalian enzyme. Likewise, contrary to that observed for mammalian SHMT, the catalytic activity of K251R TvSHMT was unaffected in the presence of PLP. The K251Q TvSHMT, however, was found to be inactive. These studies indicate that the active site of the parasite enzyme is distinct from its prokaryotic and eukaryotic counterparts and identify TvSHMT as a potential drug target.
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http://dx.doi.org/10.1128/EC.00249-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1694819PMC
December 2006

The TCL1 oncoprotein binds the RNase PH domains of the PNPase exoribonuclease without affecting its RNA degrading activity.

Cancer Lett 2007 Apr 28;248(2):198-210. Epub 2006 Aug 28.

Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095-1732, USA.

TCL1 is an AKT kinase coactivator that, when dysregulated, initiates mature lymphocyte malignancies in humans and transgenic mice. While TCL1 augments AKT pathway signaling, additional TCL1 interacting proteins that may contribute to cellular homeostasis or transformation are lacking. Here, an exoribonuclease, PNPase, was identified in a complex with TCL1. The AKT interaction domain on TCL1 bound either RNase PH repeat domain of PNPase without influencing its RNA degrading activity, which was compatible with predicted docking models for a TCL1-PNPase complex. Our data provide a novel protein interaction for mammalian PNPase that may impact TCL1 mediated transformation.
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http://dx.doi.org/10.1016/j.canlet.2006.07.006DOI Listing
April 2007

The 3D profile method for identifying fibril-forming segments of proteins.

Proc Natl Acad Sci U S A 2006 Mar 7;103(11):4074-8. Epub 2006 Mar 7.

Howard Hughes Medical Institute, University of California, Los Angeles, CA 90095, USA.

Based on the crystal structure of the cross-beta spine formed by the peptide NNQQNY, we have developed a computational approach for identifying those segments of amyloidogenic proteins that themselves can form amyloid-like fibrils. The approach builds on experiments showing that hexapeptides are sufficient for forming amyloid-like fibrils. Each six-residue peptide of a protein of interest is mapped onto an ensemble of templates, or 3D profile, generated from the crystal structure of the peptide NNQQNY by small displacements of one of the two intermeshed beta-sheets relative to the other. The energy of each mapping of a sequence to the profile is evaluated by using ROSETTADESIGN, and the lowest energy match for a given peptide to the template library is taken as the putative prediction. If the energy of the putative prediction is lower than a threshold value, a prediction of fibril formation is made. This method can reach an accuracy of approximately 80% with a P value of approximately 10(-12) when a conservative energy threshold is used to separate peptides that form fibrils from those that do not. We see enrichment for positive predictions in a set of fibril-forming segments of amyloid proteins, and we illustrate the method with applications to proteins of interest in amyloid research.
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http://dx.doi.org/10.1073/pnas.0511295103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1449648PMC
March 2006