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Steven J. Weintraub - One of the best experts on this subject based on the ideXlab platform.
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bcl xl Deamidation is regulated by multiple ion transporters and is intramolecularly catalyzed
Biochimica et Biophysica Acta, 2018Co-Authors: Scott R Manson, Sung Hee Jung, Steven J. WeintraubAbstract:Abstract In susceptible tumor cells, DNA-damaging antineoplastic agents induce an increase in intracellular pH during the premitochondrial stage of apoptosis. The rate of nonenzymatic Deamidation of two asparagines in the anti-apoptotic protein Bcl-xL is accelerated by this increase in pH. Deamidation of these asparagines is a signal for the degradation of Bcl-xL, which is a component of the apoptotic response to DNA damage. It has previously been shown that the increase in pH is mediated by the ion transporter Na+/H+ exchanger 1 in some cells. Here we demonstrate that one or more additional ion transporters also have a role in the regulation of Bcl-xL Deamidation in at least some tumor cell lines and fibroblasts. As a second, independent finding, we report that there are histidines in close proximity to the Bcl-xL Deamidation sites that are highly conserved in land-dwelling species and we present evidence that Deamidation of human Bcl-xL is intramolecularly catalyzed in a manner that is dependent upon these histidines. Further, we present evidence that these histidines act as a pH-sensitive switch that enhances the effect of the increase in pH on the rate of Bcl-xL Deamidation. The conservation of such histidines implies that human Bcl-xL is in essence “designed” to be deamidated, which provides further evidence that Deamidation serves as a bona fide regulatory post-translational modification of Bcl-xL.
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control of cellular bcl xl levels by Deamidation regulated degradation
PLOS Biology, 2013Co-Authors: Benjamin E. Deverman, Carlo Lapid, Scott R Manson, Jacob J Riehm, Rajeev Aurora, Kisun Kwon, Steven J. WeintraubAbstract:The cellular concentration of Bcl-xL is among the most important determinants of treatment response and overall prognosis in a broad range of tumors as well as an important determinant of the cellular response to several forms of tissue injury. We and others have previously shown that human Bcl-xL undergoes Deamidation at two asparaginyl residues and that DNA-damaging antineoplastic agents as well as other stimuli can increase the rate of Deamidation. Deamidation results in the replacement of asparginyl residues with aspartyl or isoaspartyl residues. Thus Deamidation, like phosphorylation, introduces a negative charge into proteins. Here we show that the level of human Bcl-xL is constantly modulated by Deamidation because Deamidation, like phosphorylation in other proteins, activates a conditional PEST sequence to target Bcl-xL for degradation. Additionally, we show that degradation of deamidated Bcl-xL is mediated at least in part by calpain. Notably, we present sequence and biochemical data that suggest that Deamidation has been conserved from the simplest extant metazoans through the human form of Bcl-xL, underscoring its importance in Bcl-xL regulation. Our findings strongly suggest that Deamidation-regulated Bcl-xL degradation is an important component of the cellular rheostat that determines susceptibility to DNA-damaging agents and other death stimuli.
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Chronoregulation by Asparagine Deamidation
Science Signaling, 2007Co-Authors: Steven J. Weintraub, Benjamin E. DevermanAbstract:Every asparagine in every protein undergoes nonenzymatic Deamidation to aspartate or isoaspartate at a rate determined by the surrounding protein structure and cellular environment. Under physiologic conditions, the Deamidation half-life of individual asparagines in proteins is proposed to range from less than a day to several centuries. More than 200 proteins have been shown to undergo Deamidation to a meaningful degree, and modeling predicts that hundreds more undergo Deamidation at rates that have the potential to be of biological consequence. Because Deamidation converts an asparagine into an aspartate or isoaspartate, it introduces a negative charge into a protein and results in the isomerization of a residue. Therefore, Deamidation has the potential to change protein function. Additionally, Deamidation is thought to render some proteins more susceptible to degradation. In most instances in which asparagine Deamidation has been identified in vivo, it is involved in pathology. Hence, Deamidation has been viewed primarily as a form of protein damage. However, the pervasiveness and evolutionary persistence of these unstable asparagines suggest that they may have a beneficial role. Notably, the change of even a single neighboring amino acid can have a marked effect on the rate of Deamidation of an asparagine. Therefore, the underlying rate of Deamidation of any asparagine is genetically programmable. This characteristic, combined with the wide range of Deamidation rates that can be programmed, imparts to asparagines the potential to serve as molecular timers that regulate protein function and stability.
Ralph M Riggin - One of the best experts on this subject based on the ideXlab platform.
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In Vivo Deamidation Characterization of Monoclonal Antibody by LC/MS/MS
Analytical Chemistry, 2005Co-Authors: Lihua Huang, Jirong Lu, Victor J Wroblewski, And John M Beals, Ralph M RigginAbstract:The spontaneous nonenzymatic Deamidation of glutaminyl and asparaginyl residues of peptides and proteins has been observed both in vitro and in vivo. Deamidation may change the structure and function of a peptide or protein, potentially resulting in decreased bioactivity, as well as alterations in pharmacokinetics and antigenicity of the protein pharmaceutical. Therefore, it is necessary to monitor the effect of storage and formulation conditions on Deamidation of a protein drug candidate. Of particular interest is the investigation of in vivo Deamidation mechanisms of protein drug candidates. Several methods are available to characterize the Deamidation of peptides and proteins. We present here a LC/MS/MS method used to evaluate the Deamidation of an antibody after in vivo administration. A humanized monoclonal IgG1 antibody (MAb) has several “hot spots” for spontaneous Deamidation. One site, amino acid residue Asn55 located in the CDR2 region of the heavy chain, is of particular interest since deamidati...
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in vivo Deamidation characterization of monoclonal antibody by lc ms ms
Analytical Chemistry, 2005Co-Authors: Lihua Huang, Jirong Lu, Victor J Wroblewski, And John M Beals, Ralph M RigginAbstract:The spontaneous nonenzymatic Deamidation of glutaminyl and asparaginyl residues of peptides and proteins has been observed both in vitro and in vivo. Deamidation may change the structure and function of a peptide or protein, potentially resulting in decreased bioactivity, as well as alterations in pharmacokinetics and antigenicity of the protein pharmaceutical. Therefore, it is necessary to monitor the effect of storage and formulation conditions on Deamidation of a protein drug candidate. Of particular interest is the investigation of in vivo Deamidation mechanisms of protein drug candidates. Several methods are available to characterize the Deamidation of peptides and proteins. We present here a LC/MS/MS method used to evaluate the Deamidation of an antibody after in vivo administration. A humanized monoclonal IgG1 antibody (MAb) has several “hot spots” for spontaneous Deamidation. One site, amino acid residue Asn55 located in the CDR2 region of the heavy chain, is of particular interest since deamidati...
Elizabeth M. Topp - One of the best experts on this subject based on the ideXlab platform.
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effect of protein structure on Deamidation rate in the fc fragment of an igg1 monoclonal antibody
Protein Science, 2009Co-Authors: Sandipan Sinha, Elizabeth M. Topp, Lei Zhang, Shaofeng Duan, Todd D Williams, Josef Vlasak, Roxana IonescuAbstract:The effects of secondary structure on asparagine (N) Deamidation in a 22 amino acid sequence (369-GFYPSDIAVEWESNGQPENNYK-390) of the crystallizable (Fc) fragment of a human monoclonal antibody (Fc IgG1) were investigated using high-resolution ultra performance liquid chromatography with tandem mass spectrometry (UPLC/MS). Samples containing either the intact Fc IgG (∼50 kD) (“intact protein”), or corresponding synthetic peptides (“peptide”) were stored in Tris buffer at 37°C and pH 7.5 for up to forty days, then subjected to UPLC/MS analysis with high energy MS1 fragmentation. The peptide deamidated only at N382 to form the isoaspartate (isoD382) and aspartate (D382) products in the ratio of ∼4:1, with a half-life of ∼3.4 days. The succinimide intermediate (Su382) was also detected; Deamidation was not observed for the other two sites (N387 and N388) in peptide samples. The intact protein showed a 30-fold slower overall Deamidation half-life of ∼108 days to produce the isoD382 and D387 products, together with minor amounts of D382. Surprisingly, the D382 and isoD387 products were not detected in intact protein samples and, as in the peptide samples, Deamidation was not detected at N388. The results indicate that higher order structure influences both the rate of N-Deamidation and the product distribution.
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Effect of viscosity on the Deamidation rate of a model Asn‐hexapeptide
Journal of Peptide Research, 2002Co-Authors: R. Li, Hageman Mj, Elizabeth M. ToppAbstract:: The effect of viscosity on the Deamidation rate of a model Asn-containing hexapeptide (l-Val-l-Tyr-Pro-l-Asn-Gly-l-Ala) was assessed in aqueous solution and in solids containing varying amounts of poly(vinyl pyrrolidone) (PVP) and water. Stability studies were conducted at 0.1 mg/mL peptide and 0–50% PVP (w/w) in aqueous solution, and at 5% (w/w) peptide and different relative humidities (31.6, 53.1, 74.4 and 96%) in the solid state. The parent peptide and its Deamidation products were analysed by reverse-phase high-performance liquid chromatography. Deamidation rates decreased with increasing solvent viscosity in a manner described by a semi-empirical mathematical model developed to describe this relationship. The results suggest that the motion of the Asn side-chain along the reaction coordinate is a function of the macroscopic solvent viscosity. However, the apparent energy barrier for the diffusive movement of the side-chain appears to be less than the energy barrier for that associated with macroscopic viscosity. The dependence of the Deamidation rate on viscosity in both viscous solution and hydrated solids further demonstrates the importance of mobility in peptide Deamidation.
Ronald T Borchardt - One of the best experts on this subject based on the ideXlab platform.
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formulation considerations for proteins susceptible to asparagine Deamidation and aspartate isomerization
Journal of Pharmaceutical Sciences, 2006Co-Authors: Aditya A Wakankar, Ronald T BorchardtAbstract:The asparagine (Asn) Deamidation and aspartate (Asp) isomerization reactions are nonenzymatic intra-molecular reactions occurring in peptides and proteins that are a source of major stability concern in the formulation of these biomolecules. The mechanisms for the Deamidation and isomerization reactions are similar since they both proceed through an intra-molecular cyclic imide (Asu) intermediate. The formation of the Asu intermediate, which involves the attack by nitrogen of the peptide backbone on the carbonyl carbon of the Asn or the Asp side chain, is the rate-limiting step in both the Deamidation and the isomerization reactions at physiological pH. In this article, the influence of factors such as formulation conditions, protein primary sequence, and protein structure on the reactivity of Asn and Asp residues in proteins are reviewed. The importance of formulation conditions such as pH and solvent dielectric in influencing Deamidation and isomerization reaction rates is addressed. Formulation strategies that could improve the stability of proteins to Deamidation and isomerization reactions are described. The review is intended to provide information to formulation scientists, based on protein sequence and structure, to predict potential degradative sites on a protein molecule and to enable formulation scientists to set appropriate formulation conditions to minimize reactivity of Asn and Asp residues in protein therapeutics.
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Chemical Pathways of Peptide Degradation. III. Effect of Primary Sequence on the Pathways of Deamidation of Asparaginyl Residues in Hexapeptides
Pharmaceutical Research, 1990Co-Authors: Kamlesh Patel, Ronald T BorchardtAbstract:Deamidation of Asn residues can occur either by direct hydrolysis of the Asn residue or via a cyclic imide intermediate. The effects of primary sequence on the pathways of Deamidation of Asn residues were studied using Val-Tyr-X-Asn-Y-Ala hexapeptides with substitution on the C-terminal side (Y) and on the N-terminal side (X) of the Asn residue. In acidic media the peptides deamidate by direct hydrolysis of the Asn residue to yield only Asp peptides, whereas under neutral or alkaline conditions, the peptides deamidate by formation of the cyclic imide intermediates which hydrolyze to yield both isoAsp and Asp peptides. At neutral to alkaline pH's the rate of Deamidation was significantly affected by the size of the amino acid on the C-terminal side of the Asn residue. The amino acid on the C-terminal side of the Asn residue has no effect on the rate of Deamidation at acidic pH. Changes in the structure of the amino acid on the N-terminal side of the Asn residue had no significant effect on the rate of Deamidation at all the pH's studied. For peptides that underwent Deamidation slowly, a reaction involving the attack of the Asn side chain on the peptide carbonyl carbon resulting in peptide bond cleavage was also observed.
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chemical pathways of peptide degradation ii kinetics of Deamidation of an asparaginyl residue in a model hexapeptide
Pharmaceutical Research, 1990Co-Authors: Kamlesh Patel, Ronald T BorchardtAbstract:Deamidation of Asn residues is a major chemical pathway of degradation of peptides and proteins. To understand better the external factors that influence Deamidation, we studied the degradation of the hexapeptide Val–Tyr–Pro–Asn–Gly–Ala, a fragment of adrenocorticotropic hormone, by HPLC. The Deamidation of this model peptide showed marked dependence on pH, temperature, and buffer composition. In the pH range 5 to 12, the peptide deamidated exclusively via a cyclic imide intermediate with the formation of both the Asp- and the isoAsp-hexapeptides. Buffer catalysis was also observed in the pH range of 7 to 11. However, at acidic pH's, the pathway of Deamidation involved direct hydrolysis of the amide side chain of Asn residue to produce only the Asp-hexapeptide.
N E Robinson - One of the best experts on this subject based on the ideXlab platform.
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prediction of primary structure Deamidation rates of asparaginyl and glutaminyl peptides through steric and catalytic effects
Journal of Peptide Research, 2004Co-Authors: N E Robinson, A B RobinsonAbstract:: The primary sequence dependence of Deamidation has been quantitatively explained on the basis of a simple steric and catalytic model. Application to the known Deamidation rates of peptides produces a table of coefficients that permits calculation of the known Deamidation rates and prediction of Deamidation rates for peptide sequences that have not yet been measured. This work permits a better understanding of Deamidation, provides a prediction procedure for protein engineering, and facilitates improved computation of peptide and protein primary, secondary, tertiary, and quaternary structure Deamidation rates.
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structure dependent nonenzymatic Deamidation of glutaminyl and asparaginyl pentapeptides
Journal of Peptide Research, 2004Co-Authors: N E Robinson, Z W Robinson, B R Robinson, A L Robinson, J A Robinson, M L Robinson, Arthur B. RobinsonAbstract:: Nonenzymatic Deamidation rates for 52 glutaminyl and 52 asparaginyl pentapeptides in pH 7.4, 37.0 °C. 0.15 m Tris-HCl buffer have been determined by direct injection mass spectrometry. These and the previously reported 306 asparginyl rates have been combined in a self-consistent model for peptide Deamidation. This model depends quantitatively upon peptide structure and involves succinimide, glutarimide and hydrolysis mechanisms. The experimental values and suitable interpolated values have been combined to provide Deamidation rate values in pH 7.4, 37.0 °C. 0.15 m Tris-HCl buffer for the entire set of 648 single-amide permutations of ordinary amino acid residues in GlyXxxAsnYyyGly and GlyXxxGlnYyyGly. Thus, knowledge about sequence-dependent Deamidation in peptides is extended to include very long Deamidation half-times in the range of 2–50 years.
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Deamidation of human proteins
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: N E Robinson, A B RobinsonAbstract:Deamidation of asparaginyl and glutaminyl residues causes time-dependent changes in charge and conformation of peptides and proteins. Quantitative and experimentally verified predictive calculations of the Deamidation rates of 1,371 asparaginyl residues in a representative collection of 126 human proteins have been performed. These rates suggest that Deamidation is a biologically relevant phenomenon in a remarkably large percentage of human proteins.
