In vitro aging of calmodulin generates isoaspartate at multiple Asn–Gly and Asp–Gly sites in calcium‐binding domains II, III, and IV

Potter, Steven M.; Henzel, William J.; Aswad, Dana W.

doi:10.1002/pro.5560021011

Cited by 75 publications

(74 citation statements)

References 54 publications

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“…Estimated values of t 1/2 of 500, 40, 60, and 500 days are used for AsnPro, AsnAsn, AsnGln, and bound Asn, respectively. The coefficients C m and C Sn were optimized by means of the D P method (23)(24)(25) with an increased set of proteins (26)(27)(28)(29). D P is a measure of the percentage accuracy in classifying the relative deamidation rates of Asn residues in a set of proteins (23).…”

Section: For Hydrogen Bondsmentioning

confidence: 99%

Deamidation of human proteins

Robinson

Robinson²

2001

Proc. Natl. Acad. Sci. U.S.A.

256

250

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Deamidation of asparaginyl and glutaminyl residues causes timedependent 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.in vivo deamidation ͉ asparaginyl residues D eamidation of asparaginyl (Asn) and glutaminyl (Gln) residues to produce aspartyl (Asp) and glutamyl (Glu) residues causes structurally and biologically important alterations in peptide and protein structures. At neutral pH, deamidation introduces a negative charge at the reaction site and can also lead to structural isomerization. Early work established that deamidation occurs in vitro and in vivo, and that the rates of deamidation depend on primary sequence, three-dimensional (3D) structure, pH, temperature, ionic strength, buffer ions, and other solution properties (1-11). It has been hypothesized (3,5,7,12,13) that Asn and Gln may serve, through deamidation, as molecular clocks which time biological processes such as protein turnover, homeostatic control, and organismic development and aging, as well as mediators of postsynthetic production of new proteins of unique biological value.Deamidation has been observed and characterized in a wide variety of proteins. It has been shown to regulate some timedependent biological processes (8, 9) and to correlate with others, such as development and aging. There are many reports of deamidation under physiological conditions in proteins of biological significance. For examples, see refs. 14-18.Extensive evidence suggests that deamidation of Asn at neutral pH usually proceeds through a cyclic imide reaction mechanism (19)(20)(21). Sometimes the Asp produced by deamidation is isomerized to isoAsp. The in vivo reversal of this isomerization has been widely reported, but reversal of deamidation itself and of the introduced negative charge has not been observed.Deamidation rates depend on the amino acid residues near Asn and Gln in the peptide chain with sequence-determined deamidation half-times at neutral pHs and 37°C in the range of 1-500 days for Asn and 100-Ͼ5,000 days for Gln (7, 13).Sequence-determined Asn and Gln deamidation rates are modulated by peptide and protein 3D structures. Deamidation of peptides is observed at both Asn and Gln, largely in accordance with sequence-controlled rates. Deamidation of proteins, which is usually slowed by 3D structure, occurs primarily at Asn except in very long-lived proteins where Gln deamidation is also observed. In a few instances, 3D structure has been reported to increase deamidation rate.The deamidation rates of individual Asn residues in a protein can be reliably predicted as a result of two recent advances. First, the sequence-controlled Asn deamidation rates of most of the 400 possible near-neighbor combinations in pentapeptide m...

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Section: For Hydrogen Bondsmentioning

confidence: 99%

Deamidation of human proteins

Robinson

Robinson²

2001

Proc. Natl. Acad. Sci. U.S.A.

256

250

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“…In cases where a peptide contains more than one Asx residue, determining the distribution of isoAsp sites can be challenging. We have used PIMTdependent radiolabeling in combination with HPLC to estimate the number of isoAsp sites within a given tryptic peptide [10,17]. The position of the isoAsp site(s) was then determined, where possible, by making use of the fact that Edman degradation is blocked at isoAsp linkages [18].…”

Section: Introductionmentioning

confidence: 99%

Selective cleavage of isoaspartyl peptide bonds by hydroxylamine after methyltransferase priming

Zhu

Aswad

2007

Analytical Biochemistry

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Formation of atypical isoaspartyl (isoAsp) sites in peptides and proteins via the deamidation-linked isomerization of asparaginyl-Xaa bonds or direct isomerization of aspartyl-Xaa bonds is a major contributor to spontaneous protein damage under mild conditions. This nonenzymatic reaction reroutes the Asx-Xaa peptide bond through the β-carbonyl of asparaginyl or aspartyl residues, thereby adding an extra carbon to the polypeptide backbone. Formation of isoAsp has been implicated in protein inactivation, aggregation, degradation, and autoimmunity. Knowing the location of isoAsp sites in proteins is important for understanding mechanisms of protein damage and for characterizing protein pharmaceuticals. Here we present a simple nonradioactive method for direct localization of isoAsp residues in peptides or proteins. Using three model peptides, we demonstrate that isoAsp linkages can be cleaved selectively and in high yield by a two-step process in which (i) the isoAsp linkage is converted into a succinimide on incubation with S-adenosyl-L-methionine and the commercially available enzyme, protein L-isoaspartyl-O-methyltransferase, and (ii) the succinimidyl bond is then cleaved by hydroxylamine under conditions that minimize cleavage of the traditional hydroxylamine-sensitive Asn-Gly and related peptide bonds. Location of the isoAsp linkage is then inferred by identifying the cleavage products by mass spectrometry or N-terminal sequencing. KeywordsCyclic imide; Hydroxylamine; Isoaspartate; IsoAsp; Protein L-isoaspartyl-O-methyltransferase; Sadenosyl-L-methionine; Succinimide Formation of isoaspartyl (isoAsp) 2 peptide bonds (also known as β-aspartyl bonds) at AsnXaa or Asp-Xaa linkages constitutes a major source of spontaneous protein damage under physiological conditions [1][2][3]. Formation of isoAsp arises from deamidation of asparaginylXaa bonds or direct isomerization of aspartyl-Xaa bonds. This process occurs via a succinimide (cyclic imide) intermediate generated when the C-flanking amide nitrogen makes a nucleophilic attack on the side-chain carbonyl group of the Asx residue (Fig. 1). Hydrolysis of the succinimide generates isoAsp and aspartyl linkages in a ratio that typically ranges from 70:30 to 85:15 [4,5]. The propensity for isoAsp formation is determined by local sequence and structural flexibility. Extensive in vitro studies indicate that sequences containing Asn-Gly, Asn-Ser, Asp-Gly, and Asn-His are most susceptible to isoAsp formation, particularly in highly flexible regions [6,7]. Under normal physiological conditions, the aberrant isoAsp linkage is converted back to a normal peptide bond by protein L-isoaspartyl-O-methyltransferase (PIMT, EC 2.1.1.77), a highly conserved and ubiquitous enzyme (Fig. 1) The overall content of isoAsp in a polypeptide can be conveniently determined by its ability to accept methyl groups from S-adenosyl-L-methionine (AdoMet) in a reaction catalyzed by the PIMT enzyme [15,16]. PIMT is available commercially as part of an isoAsp detection kit that can be used in e...

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“…Over time, such long-lived proteins are susceptible to a variety of spontaneous chemical modifications, including the deamidation of asparaginyl residues and the isomerization of aspartyl residues (7,12). These modifications arise through slow, non-enzymatic rearrangements that yield isoaspartyl residues and also some racemized aspartyl residues (13).…”

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confidence: 99%

“…Isoaspartates were found at these same positions in CaM isolated from bovine brain (17). When Ca 2ϩ was not present during the 2-week incubation, there was a dramatic increase in the isoaspartyl content of CaM, due to the deamidation of several asparaginyl residues and the isomerization of additional aspartates (6,12). Because these isoaspartates were in positions implicated in Ca 2ϩ binding, their appearance correlated with a significant loss of Ca 2ϩ -dependent regulatory activity (18).…”

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Ca2+-free Calmodulin and Calmodulin Damaged byin Vitro Aging Are Selectively Degraded by 26 S Proteasomes without Ubiquitination

Tarcsa¹,

Szymańska²,

Lecker³

et al. 2000

Journal of Biological Chemistry

104

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The ubiquitin-proteasome pathway is believed to selectively degrade post-synthetically damaged proteins in eukaryotic cells. To study this process we used calmodulin (CaM) as a substrate because of its importance in cell regulation and because it acquires isoaspartyl residues in its Ca 2؉ -binding regions both in vivo and after in vitro "aging" (incubation for 2 weeks without Ca 2؉ ). When microinjected into Xenopus oocytes, in vitro aged CaM was degraded much faster than native CaM by a proteasome-dependent process. Similarly, in HeLa cell extracts aged CaM was degraded at a higher rate, even though it was not conjugated to ubiquitin more rapidly than the native species. Ca 2؉ stimulated the ubiquitination of both species, but inhibited their degradation. Thus, for CaM, ubiquitination and proteolysis appear to be dissociated. Accordingly, purified muscle 26 S proteasomes could degrade aged CaM and native Ca 2؉ -free (apo) CaM without ubiquitination. Addition of Ca 2؉ dramatically reduced degradation of the native molecules but only slightly reduced the breakdown of the aged species. Thus, upon Ca 2؉ binding, native CaM assumes a non-degradable conformation, which most of the agedamaged species cannot assume. Thus, flexible conformations, as may arise from age-induced damage or the absence of ligands, can promote degradation directly by the proteasome without ubiquitination.

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In vitro aging of calmodulin generates isoaspartate at multiple Asn–Gly and Asp–Gly sites in calcium‐binding domains II, III, and IV

Cited by 75 publications

References 54 publications

Deamidation of human proteins

Deamidation of human proteins

Selective cleavage of isoaspartyl peptide bonds by hydroxylamine after methyltransferase priming

Ca2+-free Calmodulin and Calmodulin Damaged byin Vitro Aging Are Selectively Degraded by 26 S Proteasomes without Ubiquitination

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