RESUMO
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.
Assuntos
Antígenos CD28/imunologia , Antígenos CD8/imunologia , Imunoterapia Adotiva/métodos , Leucemia Linfocítica Crônica de Células B/terapia , Receptores de Antígenos de Linfócitos T/imunologia , Anticorpos de Cadeia Única/genética , Linfócitos T/imunologia , Sequência de Aminoácidos , Animais , Antígenos CD19/genética , Antígenos CD19/imunologia , Antígenos CD28/genética , Antígenos CD8/genética , Linhagem Celular Tumoral , Expressão Gênica , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/imunologia , Leucemia Linfocítica Crônica de Células B/patologia , Transfusão de Linfócitos , Camundongos , Plasmídeos/química , Plasmídeos/metabolismo , Domínios Proteicos , Receptores de Antígenos de Linfócitos T/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Anticorpos de Cadeia Única/química , Análise de Sobrevida , Linfócitos T/citologia , Linfócitos T/transplante , Transdução Genética , Carga Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Prion represents a unique class of pathogens devoid of nucleic acid. The deadly diseases transmitted by it between members of one species and, in certain instances, to members of other species present a public health concern. Transmissibility and the barriers to transmission between species have been suggested to arise from the degree to which a pathological protein conformation from an individual of one species can seed a pathological conformation in another species. However, this hypothesis has never been illustrated at an atomic level. Here we present three X-ray atomic structures of the same segment from human, mouse, and hamster PrP, which is critical for forming amyloid and confers species specificity in PrP seeding experiments. The structures reveal that different sequences encode different steric zippers and suggest that the degree of dissimilarity of these zipper structures gives rise to transmission barriers in prion disease, such as those that protect humans from acquiring bovine spongiform encephalopathy (BSE) and chronic wasting disease (CWD).
Assuntos
Doenças Priônicas/prevenção & controle , Doenças Priônicas/transmissão , Príons/química , Motivos de Aminoácidos , Amiloide/química , Animais , Cricetinae , Cristalografia por Raios X , Humanos , Camundongos , Fragmentos de Peptídeos/química , Conformação Proteica , Redobramento de Proteína , Proteínas Recombinantes/química , Especificidade da EspécieRESUMO
In prion inheritance and transmission, strains are phenotypic variants encoded by protein 'conformations'. However, it is unclear how a protein conformation can be stable enough to endure transmission between cells or organisms. Here we describe new polymorphic crystal structures of segments of prion and other amyloid proteins, which offer two structural mechanisms for the encoding of prion strains. In packing polymorphism, prion strains are encoded by alternative packing arrangements (polymorphs) of beta-sheets formed by the same segment of a protein; in segmental polymorphism, prion strains are encoded by distinct beta-sheets built from different segments of a protein. Both forms of polymorphism can produce enduring conformations capable of encoding strains. These molecular mechanisms for transfer of protein-encoded information into prion strains share features with the familiar mechanism for transfer of nucleic acid-encoded information into microbial strains, including sequence specificity and recognition by noncovalent bonds.
Assuntos
Amiloide/química , Príons/química , Sequência de Aminoácidos , Amiloide/ultraestrutura , Animais , Cristalografia por Raios X , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas , Camundongos , Microscopia Eletrônica , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de ProteínaRESUMO
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.
Assuntos
Amiloide/química , Insulina/metabolismo , Sequência de Aminoácidos , Amiloide/genética , Amiloide/metabolismo , Sítios de Ligação , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Cristalografia por Raios X , Diabetes Mellitus Tipo 2/metabolismo , Humanos , Insulina/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas , Proteínas Ligantes de Maltose , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ligação Proteica , Multimerização Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
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.
Assuntos
Amiloide/química , Ilhotas Pancreáticas/química , Sequência de Aminoácidos , Amiloide/genética , Amiloide/isolamento & purificação , Amiloide/metabolismo , Amiloide/ultraestrutura , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Simulação por Computador , Cristalização , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/fisiopatologia , Dissulfetos/química , Histidina/metabolismo , Humanos , Ligação de Hidrogênio , Polipeptídeo Amiloide das Ilhotas Pancreáticas , Proteínas Ligantes de Maltose , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Solubilidade , Difração de Raios XRESUMO
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.
Assuntos
Amiloide/química , Fragmentos de Peptídeos/química , Sequência de Aminoácidos , Cristalização , Modelos Moleculares , Príons/química , Conformação ProteicaRESUMO
The Mre11 complex (in Saccharomyces cerevisiae: Mre11, Rad50 and Xrs2) influences multiple facets of chromosome break metabolism. A conserved feature of the Mre11 complex is a zinc-coordinating motif in Rad50 called the Rad50 hook. We established a diploid yeast strain, rad50(hook), in which Rad50 is encoded in halves, one from each of the two RAD50 alleles, with the residues constituting the hook deleted. In all respects, rad50(hook) phenocopies complete Rad50 deficiency. Replacing the hook domain with a ligand-inducible FKBP dimerization cassette partially mitigated all phenotypes in a ligand-dependent manner. The data indicate that the Rad50 hook is critical for Mre11 complex-dependent DNA repair, telomere maintenance and meiotic double-strand break formation. Sister chromatid cohesion was unaffected by Rad50 deficiency, suggesting that molecular bridging required for recombinational DNA repair is qualitatively distinct from cohesin-mediated sister chromatid cohesion.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Divisão Celular , DNA Fúngico/genética , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/genética , Ligantes , Meiose/genética , Mutação/genética , Fenótipo , Ligação Proteica , Estrutura Terciária de Proteína , Recombinação Genética/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Telômero/metabolismoRESUMO
Recent structural studies of epidermal growth factor receptor (EGFR) family extracellular regions have identified an unexpected mechanism for ligand-induced receptor dimerization that has important implications for activation and inhibition of these receptors. Here we describe the 2.8 angstroms resolution X-ray crystal structure of the antigen binding (Fab) fragment from cetuximab (Erbitux), an inhibitory anti-EGFR antibody, in complex with the soluble extracellular region of EGFR (sEGFR). The sEGFR is in the characteristic "autoinhibited" or "tethered" inactive configuration. Cetuximab interacts exclusively with domain III of sEGFR, partially occluding the ligand binding region on this domain and sterically preventing the receptor from adopting the extended conformation required for dimerization. We suggest that both these effects contribute to potent inhibition of EGFR activation.