Carbamylation of Cysteine: A Potential Artifact in Peptide Mapping of Hemoglobins in the Presence of Urea

Lippincott, Julie; Apostoł, Izydor

doi:10.1006/abio.1998.2970

Cited by 81 publications

(57 citation statements)

References 17 publications

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“…For the purpose of product characterization, results from peptide mapping need to be interpreted with caution as artifacts during lengthy digestion could occur and lead to incorrect conclusions. [7][8][9][10][11] Another important issue to consider is that any correlation between modifications of different sites can be lost after the protein is cleaved into small peptides.…”

Section: Introductionmentioning

confidence: 99%

A new tool for monoclonal antibody analysis

Zhang

Mueller

et al. 2014

mAbs

131

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Section: Introductionmentioning

confidence: 99%

A new tool for monoclonal antibody analysis

Zhang

Mueller

et al. 2014

mAbs

131

View full text Add to dashboard Cite

“…17,20,21 R-Amino groups of alanine, cysteine, and glycine have also been reported as possible carbamylation sites. [22][23][24] To the best of our knowledge, the present communication represents the first experimental report on carbamylation of N-terminal proline in protein. It is interesting to note that the logarithm of the rate constants for carbamylation reaction of cyanate with a series of peptides has been found linearly related to the pK a values of the amino groups.…”

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confidence: 96%

Carbamylation of N-Terminal Proline

Olajuyigbe

Demitri²,

Ajele

et al. 2010

ACS Med. Chem. Lett.

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Protein carbamylation is of great concern both in vivo and in vitro. Here, we report the first structural characterization of a protein carbamylated at the N-terminal proline. The unexpected carbamylation of the R-amino group of the least reactive codified amino acid has been detected in high-resolution electron density maps of a new crystal form of the HIV-1 protease/saquinavir complex. The carbamyl group is found coplanar to the proline ring with a trans conformation. The reaction of N-terminal with cyanate ion derived from the chaotropic agent urea was confirmed by mass spectra analysis on protease single crystals. Implications of carbamylation process in vitro and in vivo are discussed.KEYWORDS Carbamylation, N-terminal proline, HIV-1 protease/saquinavir complex, single crystals T he HIV protease (PR) is an aspartic protease that shares sequence homology around the active site with other retroviral proteases, as the conserved Asp-ThrGly catalytic triad. 1 PR is required for proteolytic cleavage of viral Gag and Gag-Pol polyproteins into individual structural and functional proteins during viral maturation. 2 In the absence of this proteolysis, immature noninfectious virions are produced, thus making PR a prime target for structureassisted drug design in antiviral therapy. 3,4 The structure-assisted drug design and discovery process utilizes techniques such as protein crystallography, nuclear magnetic resonance (NMR), and computational biochemistry to guide synthesis of potential drugs. PR has been widely characterized biochemically and structurally, which has led to the discovery of HIV protease inhibitors (PIs). Their utilization in highly active antiretroviral therapy (HAART) has been a major turning point in the management of HIV/acquired immune-deficiency syndrome (AIDS). 5 Recently, we have demonstrated that high-resolution X-ray crystallography can be used as a powerful method to identify the most potent PR inhibitor present in an epimeric mixture. 6 The pseudosymmetric peptidomimetic inhibitor based on a novel Phe-Pro isostere core matched the 2-fold symmetry of the homodimeric structure of the PR. Fourier maps obtained by high-resolution diffraction data (1.3 Å) clearly showed the catalytic site fully occupied by a single ordered stereoisomer. On the contrary, several X-ray crystal structures of PR complexed with more asymmetric FDAapproved inhibitors, like darunavir, nelfinavir, and saquinavir (SQV), have the catalytic site occupied by the inhibitor oriented in two almost equivalent positions related by a pseudo-2-fold symmetry. [7][8][9][10][11][12] To investigate this order/disorder phenomenon and its possible relationship with crystal packing, we have undertaken a systematic search for new crystal forms of wild-type PR in complex with SQV. Single crystals of the PR/SQV complex were obtained by a vapor diffusion technique and analyzed by X-ray diffraction. These crystals belong to a new monoclinic crystal form, which contains two dimers of the complex in the asymmetric unit. Other crystal...

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“…Since many mRNAs that encode factors regulating cell growth and division, apoptosis, angiogenesis, and metastasis contain AREs, this mRNA-stabilizing role may thus represent a novel mechanism for the protumorigenic properties of constitutive Hsp70 overexpression. Supporting this hypothesis, we tested the ability of Hsp70 to bind and regulate the decay kinetics of two ARE-containing mRNAs that encode potent tumor-promoting proteins, vascular endothelial growth factor (VEGF) and cyclooxygenase-2 (Cox-2), tested whether these activities required the protein chaperone function of Hsp70, andtals (American Bioanalytical) and deionized by gently mixing with Amberlite MB-150 mixed-bed exchanger (1%, wt/vol; Sigma) at room temperature for 1 h to remove cyanate breakdown products that can carbamylate proteins (35). After cellular debris was pelleted (20,000 ϫ g for 30 min), His 6 -Hsp70 was purified by Ni 2ϩ affinity chromatography over a HiTrap chelating affinity column (GE Healthcare) equilibrated in column wash buffer containing 8 M urea.…”

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confidence: 99%

Hsp70 Is a Novel Posttranscriptional Regulator of Gene Expression That Binds and Stabilizes Selected mRNAs Containing AU-Rich Elements

Kishor

Tandukar

et al. 2013

Molecular and Cellular Biology

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The AU-rich elements (AREs) encoded within many mRNA 3= untranslated regions (3=UTRs) are targets for factors that control transcript longevity and translational efficiency. Hsp70, best known as a protein chaperone with well-defined peptide-refolding properties, is known to interact with ARE-like RNA substrates in vitro. Here, we show that cofactor-free preparations of Hsp70 form direct, high-affinity complexes with ARE substrates based on specific recognition of U-rich sequences by both the ATP-and peptide-binding domains. Suppressing Hsp70 in HeLa cells destabilized an ARE reporter mRNA, indicating a novel ARE-directed mRNA-stabilizing role for this protein. Hsp70 also bound and stabilized endogenous ARE-containing mRNAs encoding vascular endothelial growth factor (VEGF) and Cox-2, which involved a mechanism that was unaffected by an inhibitor of its protein chaperone function. Hsp70 recognition and stabilization of VEGF mRNA was mediated by an ARE-like sequence in the proximal 3=UTR. Finally, stabilization of VEGF mRNA coincided with the accumulation of Hsp70 protein in HL60 promyelocytic leukemia cells recovering from acute thermal stress. We propose that the binding and stabilization of selected ARE-containing mRNAs may contribute to the cytoprotective effects of Hsp70 following cellular stress but may also provide a novel mechanism linking constitutively elevated Hsp70 expression to the development of aggressive neoplastic phenotypes.

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Carbamylation of Cysteine: A Potential Artifact in Peptide Mapping of Hemoglobins in the Presence of Urea

Cited by 81 publications

References 17 publications

A new tool for monoclonal antibody analysis

A new tool for monoclonal antibody analysis

Carbamylation of N-Terminal Proline

Hsp70 Is a Novel Posttranscriptional Regulator of Gene Expression That Binds and Stabilizes Selected mRNAs Containing AU-Rich Elements

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