The tubular gland of the chicken oviduct is an attractive system for protein expression as large quantities of proteins are deposited in the egg, the production of eggs is easily scalable and good manufacturing practices for therapeutics from eggs have been established. Here we examined the ability of upstream and downstream DNA sequences of ovalbumin, a protein produced exclusively in very high quantities in chicken egg white, to drive tissue-specific expression of human mAb in chicken eggs. To accommodate these large regulatory regions, we established and transfected lines of chicken embryonic stem (cES) cells and formed chimeras that express mAb from cES cell-derived tubular gland cells. Eggs from high-grade chimeras contained up to 3 mg of mAb that possesses enhanced antibody-dependent cellular cytotoxicity (ADCC), nonantigenic glycosylation, acceptable half-life, excellent antigen recognition and good rates of internalization.

The fatty acid synthases (FASs) 1 associated with the soluble cytoplasm of yeast and animal cells comprise large multifunctional polypeptides that contain all of the catalytic components required for the synthesis of long-chain fatty acids from malonyl-CoA de novo. These multifunctional polypeptides are commonly referred to as type I FASs. The animal FASs consist of two identical polypeptides of approximately 2500 residues (␣ 2 ), whereas the yeast FAS comprises six copies each of two nonidentical polypeptides (␣ 6 ␤ 6 ; ␣ ϭ 1845, ␤ ϭ 1887 residues) (1, 2). In most bacteria and in plant plastids, the various catalytic components of the FAS are present on separate discrete polypeptides and are commonly referred to as type II FASs (3). An essential component of both type I and type II systems is a small molecular mass domain/polypeptide known as an acyl carrier protein (ACP) that is posttranslationally modified, by insertion of a 20-Å-long phosphopantetheinyl moiety, derived from CoA, to a positionally conserved serine residue (4). The terminal sulfhydryl of the phosphopantetheinyl moiety provides the site of covalent attachment of the substrates and the growing fatty acyl chain, so that the phosphopantetheine plays an essential role as a "swinging arm" in the translocation of intermediates between different catalytic sites of the FASs (5). ACPs fulfill a similar role in the type I and type II polyketide synthases (PKSs) found mainly in bacteria and fungi that are capable of elaborating a broad range of secondary metabolites.Fungi (6, 7), animal, and plant cells also contain a type II FAS system in their mitochondria. The role of the mitochondrial FASs is not well established, but it has been suggested that, at least in fungi and plants, they may serve to provide octanoate, the precursor of lipoic acid and/or long-chain fatty acids that are used in the remodeling of mitochondrial phospholipids (7-10). The ACP component of the mitochondrial FAS appears to be associated with the respiratory complex I in animals and in Neurospora crassa (11,12). Phosphopantetheinylated carrier proteins also play an essential role as components of the non-ribosomal peptide synthases found in microorganisms that are responsible for producing a variety of short peptides containing both proteogenic and unusual amino acids (13). The nonribosomal peptide synthases also comprise multifunctional polypeptides in which the role of the carrier protein domain is to translocate amino acyl moieties from an adenylation domain to a condensation domain, where formation of a new peptide bond takes place (14).Enzymes capable of phosphopantetheinylating carrier proteins involved in the biosynthesis of fatty acids, polyketides, and peptides have been identified and characterized from a variety of sources. Many organisms have more than one phosphopantetheine transferase (PPTase), and different PPTases commonly are utilized to service carrier proteins associated with FASs and non-ribosomal peptide synthases within the same species (4, 15). Yeast utilizes...

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Rational modulation of the immune response with biologics represents one of the most promising and active areas for the realization of new therapeutic strategies. In particular, the use of function blocking monoclonal antibodies targeting checkpoint inhibitors such as CTLA-4 and PD-1 have proven to be highly effective for the systemic activation of the human immune system to treat a wide range of cancers. Ipilimumab is a fully human antibody targeting CTLA-4 that received FDA approval for the treatment of metastatic melanoma in 2011. Ipilimumab is the first-in-class immunotherapeutic for blockade of CTLA-4 and significantly benefits overall survival of patients with metastatic melanoma. Understanding the chemical and physical determinants recognized by these mAbs provides direct insight into the mechanisms of pathway blockade, the organization of the antigen-antibody complexes at the cell surface, and opportunities to further engineer affinity and selectivity. Here, we report the 3.0 Å resolution X-ray crystal structure of the complex formed by ipilimumab with its human CTLA-4 target. This structure reveals that ipilimumab contacts the front β-sheet of CTLA-4 and intersects with the CTLA-4:Β7 recognition surface, indicating that direct steric overlap between ipilimumab and the B7 ligands is a major mechanistic contributor to ipilimumab function. The crystallographically observed binding interface was confirmed by a comprehensive cell-based binding assay against a library of CTLA-4 mutants and by direct biochemical approaches. This structure also highlights determinants responsible for the selectivity exhibited by ipilimumab toward CTLA-4 relative to the homologous and functionally related CD28.immunotherapy | X-ray crystallography | CTLA-4 | ipilimumab | cancer

In Alzheimer’s disease (AD), an extensive accumulation of extracellular amyloid plaques and intraneuronal tau tangles, along with neuronal loss, is evident in distinct brain regions. Staging of tau pathology by postmortem analysis of AD subjects suggests a sequence of initiation and subsequent spread of neurofibrillary tau tangles along defined brain anatomical pathways. Further, the severity of cognitive deficits correlates with the degree and extent of tau pathology. In this study, we demonstrate that phospho-tau (p-tau) antibodies, PHF6 and PHF13, can prevent the induction of tau pathology in primary neuron cultures. The impact of passive immunotherapy on the formation and spread of tau pathology, as well as functional deficits, was subsequently evaluated with these antibodies in two distinct transgenic mouse tauopathy models. The rTg4510 transgenic mouse is characterized by inducible over-expression of P301L mutant tau, and exhibits robust age-dependent brain tau pathology. Systemic treatment with PHF6 and PHF13 from 3 to 6 months of age led to a significant decline in brain and CSF p-tau levels. In a second model, injection of preformed tau fibrils (PFFs) comprised of recombinant tau protein encompassing the microtubule-repeat domains into the cortex and hippocampus of young P301S mutant tau over-expressing mice (PS19) led to robust tau pathology on the ipsilateral side with evidence of spread to distant sites, including the contralateral hippocampus and bilateral entorhinal cortex 4 weeks post-injection. Systemic treatment with PHF13 led to a significant decline in the spread of tau pathology in this model. The reduction in tau species after p-tau antibody treatment was associated with an improvement in novel-object recognition memory test in both models. These studies provide evidence supporting the use of tau immunotherapy as a potential treatment option for AD and other tauopathies.

A HIV-1 Nef affinity column was used to purify a 35-kDa Nef-interacting protein from T-cell lysates. All of these mutations also abrogated the ability of Nef to down-regulate CD4 from the surface of HIV-infected cells. Based on the x-ray and NMR structures of Nef, these residues define a surface on Nef critical for CD4 down-regulation. A subset of these mutations also affected the ability of Nef to down-regulate major histocompatibility complex class I. These results, taken together with previous studies, identify a region on Nef critical for most of its known functions. However, not all Nef alleles bind to hTE with high affinity, so the role of hTE during HIV infection remains uncertain.

The structural basis for the dual specificity of the malonyl-CoA/acetyl-CoA:acyl carrier protein S-acyltransferase associated with the multifunctional animal fatty acid synthase has been investigated by mutagenesis. Arginine 606, which is positionally conserved in the transacylase domains of all multifunctional fatty acid and polyketide synthases, was replaced by alanine or lysine in the context of the isolated transacylase domain, and the mutant proteins were expressed in Escherichia coli. Malonyl transacylase activity of the Arg-606 3 Ala and Arg-606 3 Lys mutant enzymes was reduced by 100-and 10-fold, respectively. In contrast, acetyl transacylase activity was increased 6.6-fold in the Arg-606 3 Ala mutant and 1.7-fold in the Arg-606 3 Lys mutant. Kinetic studies revealed that selectivity of the enzyme for acetyl-CoA was increased >16,000-fold by the Ala mutation and 16-fold by the Lys mutation. Activity toward medium chain length acyl thioesters was also increased >3 orders of magnitude by mutation of Arg-606, so that the Ala-606 enzyme is an effective medium chain length fatty acyl transacylase. These results indicate that Arg-606 plays an important role in the binding of malonyl moieties to the transacylase domain but is not required for binding of acetyl moieties; these results are also consistent with a mechanism whereby interaction between the positively charged guanidinium group of Arg-606 and the free carboxylate anion of the malonyl moiety serves to position this substrate in the active site of the enzyme.The animal FAS 1 consists of two identical polypeptides, each carrying six enzymes and an acyl carrier protein, that are juxtaposed to form two centers for the synthesis of palmitic acid from acetyl-and malonyl-CoA (1-3). The iterative condensation of an acetyl moiety with successive malonyl moieties and reduction of the ␤-keto intermediates normally results in the formation of palmitic acid as the major product. Initiation of the series of condensation reactions necessitates the translocation of an acetyl moiety and subsequently seven malonyl moieties, from CoA thioester to the 4Ј-phosphopantetheine of the acyl carrier protein domain of the FAS. A single transacylase enzyme is responsible for translocation of both substrates, and intermediate formation of acyl-O-serine intermediates occurs at the same site (4). The employment of a common enzyme for the loading of both substrates has important consequences in terms of the kinetics of the FAS reaction sequence, since substrate binding to the transacylase domain is random and each substrate is a competitive inhibitor for the other. Thus, for synthesis to proceed efficiently, inappropriately bound substrates must be rapidly removed by transfer back to the CoA acceptor (5). This substrate sorting or "self-editing" process occurs extremely rapidly, with turnover numbers Ͼ100 s Ϫ1 , and does not limit the rate of acyl chain assembly, provided an appropriate concentration of free CoA is available (5, 6).The chain initiation mechanism for FASs that consist ...

The antagonistic effect of cAMP on the insulin-induced expression of fatty acid synthase (FAS) in liver could be mimicked in vitro using H4IIE hepatoma cells, both by measuring the response of the endogenous FAS gene and by assaying expression of transfected reporter genes containing promoter elements of the FAS gene. 5-Deletion analysis and replacement mutagenesis revealed that an essential element required for cAMP antagonism of the insulin effect is an inverted CCAAT box located between nucleotides ؊99 and ؊92. DNase I footprinting and gel shift analysis revealed that this region can bind a protein present in nuclei of liver and spleen, organs that express high and undetectable levels of FAS, respectively. This protein is not a CCAAT/enhancerbinding protein, C/EBP. Thus, the FAS gene appears unusual in that the sequence element required for transcriptional regulation by cAMP is neither a cAMP response element (CRE) nor a binding site for AP-1, AP-2, or C/EBP. These results suggest that essential to the regulation of FAS transcription by cAMP is the interaction of an inverted CCAAT box motif with a constitutively produced trans-acting factor that either itself undergoes modification in response to cAMP or associates with a protein that is produced or modified by cAMP exposure.It has long been recognized that glycogen metabolism, gluconeogenesis, glycolysis, fatty acid oxidation, and synthesis are all coordinately regulated in the liver by short-term regulatory mechanisms involving primarily allosteric and covalent modulation of key enzymes (1). Critical to this short-term regulation are the opposing actions of insulin and glucagon. Glucagon, via its intracellular messenger, cAMP, activates protein kinases involved in the phosphorylation of key enzymes and transforms the liver from a glycogenic, glycolytic, lipogenic tissue to a glycogenolytic, gluconeogenic, and fatty acid-oxidizing tissue. Initially, when the food supply is stopped, serum insulin concentration falls and glucagon concentration rises, triggering this short-term regulatory mechanism. On refeeding, insulin concentration rises, glucagon falls, and these changes are reversed, and the liver reverts to its role as a glycogenic, glycolytic, lipogenic tissue. It seems likely that antagonism between insulin and glucagon may also serve in long-term regulation of these pathways by controlling transcription of key genes (2).For example, on prolonged fasting, cAMP may eventually initiate the down-regulation of transcription of lipogenic enzymes. Indeed, it has been shown that injection of dibutyryl cAMP will prevent the activation of FAS 1 transcription in the liver that normally accompanies refeeding (3).All of the long-term changes in lipogenesis that occur in response to dietary, hormonal, and developmental cues appear to be accompanied by changes in the rate of transcription of the FAS gene in a tissue-specific manner. Elucidation of the sequence of the entire rat FAS gene, including 6.1 kilobases of the 5Ј-flanking region (4, 5), has made possible deta...