Modification Of Lysine Side Chains Research Articles

Structures of prokaryotic ubiquitin-like protein Pup in complex with depupylase Dop reveal the mechanism of catalytic phosphate formation

Pupylation is the post-translational modification of lysine side chains with prokaryotic ubiquitin-like protein (Pup) that targets proteins for proteasomal degradation in mycobacteria and other members of Actinobacteria. Pup ligase PafA and depupylase Dop are the two enzymes acting in this pathway. Although they share close structural and sequence homology indicative of a common evolutionary origin, they catalyze opposing reactions. Here, we report a series of high-resolution crystal structures of Dop in different functional states along the reaction pathway, including Pup-bound states in distinct conformations. In combination with biochemical analysis, the structures explain the role of the C-terminal residue of Pup in ATP hydrolysis, the process that generates the catalytic phosphate in the active site, and suggest a role for the Dop-loop as an allosteric sensor for Pup-binding and ATP cleavage.

Drug-Mimicking Nanofibrous Peptide Hydrogel for Inhibition of Inducible Nitric Oxide Synthase.

In this work, we develop a drug-mimicking nanofibrous peptide hydrogel that shows long-term bioactivity comparable to a small-molecule inhibitor of inducible nitric oxide synthase (iNOS). The iNOS inhibitor, N 6-(1-iminoethyl)-l-lysine (l-NIL), is a positively charged amino acid whose structure could be readily integrated into the framework of a positively charged multidomain peptide (MDP) through the modification of lysine side chains. This new l-NIL-MDP maintains the self-assembling properties of the base peptide, forming β-sheet nanofibers, which entangle into a thixotropic hydrogel. The l-NIL-MDP hydrogel supports cell growth in vitro and allows syringe-directed delivery that persists in a targeted location in vivo for several weeks. Multiple characterization assays demonstrate the bioactivity of the l-NIL-MDP hydrogel to be comparable to the l-NIL small molecule. This includes iNOS inhibition of macrophages in vitro, reduced nitrotyrosine immunostaining in murine subcutaneous histology, and reduced serum levels of vascular endothelial growth factor in vivo. This study expands the toolbox of available peptide hydrogel scaffold designs that can modify biological activity without the need for any additional small-molecule drugs, proteins, or cells.

Artificial Modification of an Enzyme for Construction of Cross-Reactive Polyion Complexes To Fingerprint Signatures of Proteins and Mammalian Cells.

A novel strategy for construction of cross-reactive enzyme-based sensors have been developed based on chemical modification of lysine ε-NH3(+) groups in β-galactosidase (β-Gal) from Aspergillus oryzae with various acid anhydrides. Modification of lysine side chains markedly altered the kinetic parameters of β-Gal (KM and kcat), whereas catalytic activity remained and the tertiary structure was maintained for all modified β-Gals. The addition of cationic PEGylated polymers to reactive solutions caused the formation of polyion complexes (PICs) with marked inhibition of the modified β-Gal activity. Therefore, the obtained PICs can be used to construct cross-reactive enzyme-based sensors without any purification. With addition of each analyte protein or mammalian cell to the PIC library, the modified β-Gals were partially released from PICs, and therefore the catalytic activities showed characteristic recovery fingerprints. Linear discriminant analysis (LDA) of fingerprints generated by the combination of only three PICs enabled successful discrimination of 0.5 μg/mL human plasma proteins (albumin, immunoglobulin G, transferrin, fibrinogen, α1-antitrypsin, C-reactive protein), and semiquantification of inflammatory biomarker proteins in buffer (0.3-8.1 μg/mL) and human serum (2-100 μg/mL) with comparable sensitivity for diagnosis in human blood samples. Moreover, we identified five mammalian (human, bovine, fetal bovine, horse, and rabbit) sera containing 2.5 μg/mL serum proteins, and three human cancer cell lines [A549 (lung), MG63 (bone), HuH7 (liver)] and a human mesenchymal stem cell line (UE7T-13) (1500 cells/mL) with 100% accuracy.

Pupylation-dependent and -independent proteasomal degradation in mycobacteria.

Bacteria make use of compartmentalizing protease complexes, similar in architecture but not homologous to the eukaryotic proteasome, for the selective and processive removal of proteins. Mycobacteria as members of the actinobacteria harbor proteasomes in addition to the canonical bacterial degradation complexes. Mycobacterial proteasomal degradation, although not essential during normal growth, becomes critical for survival under particular environmental conditions, like, for example, during persistence of the pathogenic Mycobacterium tuberculosis in host macrophages or of environmental mycobacteria under starvation. Recruitment of protein substrates for proteasomal degradation is usually mediated by pupylation, the post-translational modification of lysine side chains with the prokaryotic ubiquitin-like protein Pup. This substrate recruitment strategy is functionally reminiscent of ubiquitination in eukaryotes, but is the result of convergent evolution, relying on chemically and structurally distinct enzymes. Pupylated substrates are recognized by the ATP-dependent proteasomal regulator Mpa that associates with the 20S proteasome core. A pupylation-independent proteasome degradation pathway has recently been discovered that is mediated by the ATP-independent bacterial proteasome activator Bpa (also referred to as PafE), and that appears to play a role under stress conditions. In this review, mechanistic principles of bacterial proteasomal degradation are discussed and compared with functionally related elements of the eukaryotic ubiquitin-proteasome system. Special attention is given to an understanding on the molecular level based on structural and biochemical analysis. Wherever available, discussion of in vivo studies is included to highlight the biological significance of this unusual bacterial degradation pathway.

Does electron capture dissociation (ECD) provide quantitative information on the chemical modification of lysine side chains in proteins? The glycation of ubiquitin.

Electron capture dissociation (ECD) as a method of quantitative and qualitative study of glycated ubiquitin was investigated. ECD has been successfully applied for sequencing of modified peptides and assigning glycated Lys residues. By using a hybrid Fourier transform mass spectrometry (FT-MS) system equipped for ECD, a series of multiply glycated ubiquitin ions was observed. Ions of the glycated ubiquitin with a defined number of glucose moieties attached to the protein were isolated by quadrupol and fragmented in the ICR cell by the ECD method. The fragmentation spectrum was dominated by c(n) and (z+1)n ions. The ECD technique was tested for the quantitative analysis of the modified ubiquitin and isomeric glycated peptides (fragments of bovine serum albumin (BSA)). Obtained results indicate that the ECD fragmentation cannot be applied for the quantitative determination of the relative reactivities of respective Lys residues in the ubiquitin.

Alkaline pH -dependent differential unfolding characteristics of mesophilic and thermophilic homologs of dimeric serine hydroxymethyltransferase

Environmental variables such as pH can significantly influence the folding and stability of a protein molecule. In the present investigation, we compared the alkaline pH-induced unfolding of two homologous serine hydroxymethyltransferase from mesophilic Bacillus subtilis (bsSHMT) and thermophilic Bacillus stearothermophilus (bstSHMT) using various biophysical techniques. The thermophilic enzyme bstSHMT was found to be more resistant to alkaline denaturation compared to its mesophilic counterpart, bsSHMT. Unfolding studies using domain-swapped chimera, constructed by swapping the C-terminal domain of these two wild-type proteins, revealed that C-terminal domain plays a pivotal role in the folding, stability and subunit interaction of these proteins. Primary amino acid sequence analysis of the proteins showed that bsSHMT has six unconserved lysine residues in C-terminal domain, which are absent in bstSHMT. Chemical modification of lysine side chains resulted in stabilization of monomers, only in case of bsSHMT. Moreover, comparison between homology model of bsSHMT with the crystal structure of bstSHMT revealed that a small stretch of 11 amino acids at the end of C-terminal domain was found protruding outside the molecule as a flexible coiled structure in bsSHMT. Taken together these findings suggest that possibly the presence of these non-identical lysine moieties and a small extension of C-terminal domain may be responsible for low stability of bsSHMT under alkaline pH condition.