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Autophagy, a pathway primarily relevant for cell survival, and apoptosis, a process invariably leading to cell death, are the two main mechanisms of cellular self-destruction, which are essential in cell growth, neurodegeneration, tumor suppression, stress and immune response. Currently, a potential crosstalk between apoptosis and autophagy is subject to intensive investigations since recently some direct junctions became obvious. The respective protein-protein interaction network, however, remains to be elucidated in detail. The γ-aminobutyric acid type A (GABA A ) receptor-associated protein GABARAP belongs to a family of proteins implicated in intracellular transport events and was shown to be associated to autophagic processes. Using a phage display screening against the target protein GABARAP, we identified the proapoptotic protein Nix/Bnip3L to be a potential GABARAP ligand. In vitro binding studies, pull-down analysis, coimmunoprecipitation assays and colocalization studies confirmed a direct interaction of both proteins in mammalian cells.

BackgroundRegulatory approval for a biosimilar product is provided on the basis of its comparability to an originator product. A thorough physicochemical and functional comparability exercise is a key element in demonstrating biosimilarity. Here we report the characterization of a proposed biosimilar rituximab (GP2013) and originator rituximab.ObjectiveTo compare GP2013 with originator rituximab using an extensive array of routine analytical and extended characterization methods.MethodsPrimary and higher order protein structures were analyzed using a variety of methods that included high-performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI-MS), peptide mapping with UV and MS detection, circular dichroism (CD), Fourier transform infrared (FTIR) spectroscopy, hydrogen deuterium exchange (HDX) MS, 1D 1H nuclear magnetic resonance (NMR) spectroscopy, X-ray crystallography and differential scanning calorimetry (DSC). Charge and amino acid modifications were assessed using cation exchange chromatography (CEX) and peptide mapping using reversed-phase (RP) HPLC. Boronate affinity chromatography was used to determine the relative amount of glycation. Glycans were identified and quantified after 2-aminobenzamide (2-AB) labeling and separation using normal phase HPLC with fluorescence and MS detection, respectively. Glycan site occupancy was determined using reducing capillary electrophoresis with sodium dodecyl sulfate (CE-SDS). Size heterogeneity was determined using reducing and non-reducing CE-SDS, size exclusion chromatography (SEC) and asymmetric flow field flow fractionation (AF4). Biological characterization included a series of bioassays (in vitro target binding, antibody-dependent cell-mediated cytotoxicity [ADCC], complement-dependent cytotoxicity [CDC] and apoptosis) and surface plasmon resonance (SPR) Fc receptor binding assays.ResultsIntact mass analysis of GP2013 and the heavy and light chains using RP HPLC–ESI–MS revealed the expected molecular mass of rituximab. The amino acid sequence was shown to be identical between GP2013 and the originator rituximab. Further sequence confirmation using RP-HPLC-UV/MS peptide mapping showed non-distinguishable chromatograms for Lys-C digested GP2013 and originator rituximab. The higher order structure of GP2013 was shown to be indistinguishable from originator rituximab using a large panel of redundant and orthogonal methods. GP2013 and originator rituximab were comparable with regard to charge variants, specific amino acid modifications and the glycan pattern. GP2013 was also shown to have similar purity, aggregate and particle levels when compared with the originator. Functionally, and by using a comprehensive set of bioassays and binding assays covering a broad range of rituximab’s functional activities, GP2013 could not be distinguished from originator rituximab.ConclusionGP2013 was shown to be physicochemically highly similar to originator rituximab at the level of primary and higher order structure, post-translational modifications ...

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Hyperreactive sulfhydryl groups associated with the Ca 2؉ release protein from sarcoplasmic reticulum are shown to have a well defined reduction potential that is sensitive to the cellular environment. Ca 2؉ channel activators lower the redox potential of the ryanodine receptor, which favors the oxidation of thiols and the opening of the Ca 2؉ release protein. Recently, a great deal of attention has focused on understanding the sensitivity of the Ca 2ϩ release mechanism to cellular redox changes (3). It is well established that oxidation of critical thiol groups activates the Ca 2ϩ release mechanism, whereas addition of thiol reducing agents close down the Ca 2ϩ channel (4 -6). Oxidative modification of Ca 2ϩ channel function has been observed at the level of skinned fibers in Ca 2ϩ flux measurements in single channel measurements and at the level of high affinity ryanodine binding measurements (4 -9). Moreover, it has been observed that reactive oxygen species activate Ca 2ϩ release from SR (10 -13) and may act as redox active signaling molecules to activate Ca 2ϩ transport (14). It is clear from the above studies that redox reactions may play a critical role in controlling the kinetics of the Ca 2ϩ release mechanism. Furthermore, from experiments carried out with a fluorescent maleimide, it has been shown that the reactivity of these hyperreactive thiols is very sensitive to the concentration of Ca 2ϩ channel modulators such as Ca 2ϩ , Mg 2ϩ , and caffeine (15).The redox potential within the cell is controlled by the concentrations of reduced glutathione (GSH), oxidized glutathione (GSSG), NAD ϩ , and NADH. Zable et al. (16) have shown that GSH inhibits Ca 2ϩ channel activity and equilibrium ryanodine binding, whereas GSSG stimulates the activity of the receptor. This suggests that changes in the cellular redox potential may influence the degree of activation of the Ca 2ϩ release mechanism and effect the myoplasmic Ca 2ϩ concentration and the contractile state of muscle.Instead of using the more traditional method of measuring equilibrium ryanodine binding, in this study a model was developed in which the rate of ryanodine binding was related to the redox state of the receptor. For the first time, hyperreactive sulfhydryl groups associated with the Ca 2ϩ release mechanism from skeletal muscle SR were shown to have a well defined redox potential, and this redox potential was controlled by physiologically relevant Ca 2ϩ channel activators and inhibitors. Although it is unlikely that the redox state of these thiols controls excitation-contraction coupling, our results suggest that during oxidative stress, these hyperreactive thiols oxidize and activate the SR Ca 2ϩ release mechanism and alter the Ca 2ϩ sensitivity of the release channel. Under mild oxidative stress, relatively small changes in the cellular redox potential can contribute to significant stimulation of the ryanodine receptor.EXPERIMENTAL PROCEDURES SR vesicles were isolated from rabbit fast twitch skeletal muscle by the method of MacLennan (17) wit...

The control of neurotransmitter receptor expression and delivery to the postsynaptic membrane is of critical importance for neural signal transduction at synapses. The sorting, targeting and degradation of neurotransmitter receptors require mechanisms to regulate intracellular vesicular protein transport. These dynamic processes play a key role in the construction and functional maintenance of synapses, and are one of the underlying mechanisms of synaptic plasticity. 4-Aminobutyrate type A (GABA A ) receptor-associated protein (GABA-RAP) is a ubiquitin-like modifier implicated in the intracellular trafficking of GABA A receptors, and belongs to a family of proteins involved in intracellular vesicular transport processes, such as autophagy and intra-Golgi transport. In this article, it is demonstrated that calreticulin is a high affinity ligand of GABARAP. Calreticulin, although best known for its functions as a Ca 2+-dependent chaperone and a Ca 2+ -buffering protein in the endoplasmic reticulum, is also localized to the cytosol and exerts a variety of extra-endoplasmic reticulum functions. By phage display screening of a randomized peptide library, peptides that specifically bind GABARAP were identified. Their amino acid sequences allowed us to identify calreticulin as a potential GABARAP binding protein. GABARAP binding to calreticulin was confirmed by pull-down experiments with brain lysate and colocalization studies in N2a cells. Calreticulin and GABARAP interact with a dissociation constant K d ¼ 64 nm and a mean lifetime of the complex of 20 min. Thus, the interaction between GABARAP and calreticulin is the strongest so far reported for each protein.Abbreviations CaN, calcineurin; CNX, calnexin; CRT, calreticulin; DDX47, DEAD box polypeptide 47; ER, endoplasmic reticulum; GABA A receptor, 4-aminobutyrate type A receptor; GABARAP, GABA A receptor-associated protein; GRIP1, glutamate receptor-interacting protein 1; GST, glutathione S-transferase; Ins(1,4,5)P 3 , inositol 1,4,5-triphosphate; HSQC, heteronuclear single quantum coherence; LC3, light chain 3; MAP1 LC3, microtubule-associated protein 1 light chain 3; N2a, NEURO-2a; NHS, N-hydroxysuccinimide; NSF, N-ethylmaleimide sensitive factor; NaCl ⁄ P i , phosphate buffer pH 7.6; PRIP-1, phospholipase C-related inactive protein type 1; PSSM, position-specific scoring matrix; SPR, surface plasmon resonance; SUMO, small ubiquitin-like modifier; UBL, ubiquitin-like modifier; Ubq, ubiquitin; ULK1, unc-51-like kinase 1.

The SARS related Coronavirus genome contains a variety of novel accessory genes. One of these, called ORF7a or ORF8, code for a protein, known as 7a, U122 or X4. We set out to determine the three-dimensional structure of the soluble ectodomain of this type-I transmembrane protein by nuclear magnetic resonance spectroscopy. The fold of the protein is the first member of a further variation of the immunoglobulin like beta-sandwich fold. Because X4 does not reveal significant sequence homologies to proteins in the data bases, we carried out a structure based similarity search for proteins with known function. High structural similarity to Dl domains of ICAM-1 and ICAM-2, and common features in amino acid sequence between X4 and ICAM-1, suggest X4 to possess binding activity for the alpha(L) integrin I domain of LFA-1. Further, based on this structure based prediction, potential functions of X4 in virus replication and pathogenesis are discussed.

In contrast with conventional drugs, biopharmaceuticals are highly complex molecules with remarkable heterogeneity. Protein glycosylation is an inherent source of this heterogeneity and also affects the safety, efficacy, and serum half-life of therapeutic glycoproteins. Therefore analysis of the glycan pattern is an important issue for characterization and quality control in the biopharmaceutical industry. In this publication we describe a complete workflow for the analysis of protein N-glycans. The sample-preparation procedure, consisting of the release of the N-glycans by PNGase-F, followed by fluorescence labeling with 2-aminobenzamide and removal of excess label, was optimized to avoid alteration of the glycan sample. Subsequently, labeled glycans were analyzed by hydrophilic-interaction liquid chromatography (HILIC) with fluorescence detection. The developed method was validated for analysis of antibody N-glycans. To demonstrate the accuracy of the method an antibody sample was additionally analyzed by an orthogonal method. The antibody was digested with lysyl endopeptidase and the (glyco-)peptides were analyzed by RP-HPLC-MS. The consistency of the results between these two methods demonstrates the reliability of the glycan analysis method introduced herein.