The key role of glutamine for protein expression and isotopic labeling in insect cells.

Nuclear magnetic resonance studies of many physiologically important proteins have long been impeded by the necessity to express such proteins in isotope-labeled form in higher eukaryotic cells and the concomitant high costs of providing isotope-labeled amino acids in the growth medium. Economical routes use isotope-labeled yeast or algae extracts but still require expensive isotope-labeled glutamine. Here, we have systematically quantified the effect of 15N2-glutamine on the expression and isotope labeling of different proteins in insect cells. Sufficient levels of glutamine in the medium increase the protein expression by four to five times relative to deprived conditions. 1H-15N nuclear magnetic resonance spectroscopy shows that the 15N atoms from 15N2-glutamine are scrambled with surprisingly high (60-70%) efficiency into the three amino acids alanine, aspartate, and glutamate. This phenomenon gives direct evidence that the high energy demand of insect cells during baculovirus infection and concomitant heterologous protein expression is predominantly satisfied by glutamine feeding the tricarboxylic acid cycle. To overcome the high costs of supplementing isotope-labeled glutamine, we have developed a robust method for the large-scale synthesis of 15N2-glutamine and partially deuterated 15N2-glutamine-α,ß,ß-d3 from inexpensive precursors. An application is shown for the effective large-scale expression of the isotope-labeled ß1-adrenergic receptor using the synthesized 15N2-glutamine-α,ß,ß-d3.

The molecular basis of Abelson kinase regulation by its αI-helix.

Abelson tyrosine kinase (Abl) is regulated by the arrangement of its regulatory core, consisting sequentially of the SH3, SH2, and kinase (KD) domains, where an assembled or disassembled core corresponds to low or high kinase activity, respectively. It was recently established that binding of type II ATP site inhibitors, such as imatinib, generates a force from the KD N-lobe onto the SH3 domain and in consequence disassembles the core. Here, we demonstrate that the C-terminal αI-helix exerts an additional force toward the SH2 domain, which correlates both with kinase activity and type II inhibitor-induced disassembly. The αI-helix mutation E528K, which is responsible for the ABL1 malformation syndrome, strongly activates Abl by breaking a salt bridge with the KD C-lobe and thereby increasing the force onto the SH2 domain. In contrast, the allosteric inhibitor asciminib strongly reduces Abl's activity by fixating the αI-helix and reducing the force onto the SH2 domain. These observations are explained by a simple mechanical model of Abl activation involving forces from the KD N-lobe and the αI-helix onto the KD/SH2SH3 interface.

Changing nucleotide specificity of the DEAD-box helicase Hera abrogates communication between the Q-motif and the P-loop.

DEAD-box proteins disrupt or remodel RNA and protein/RNA complexes at the expense of ATP. The catalytic core is composed of two flexibly connected RecA-like domains. The N-terminal domain contains most of the motifs involved in nucleotide binding and serves as a minimalistic model for helicase/nucleotide interactions. A single conserved glutamine in the so-called Q-motif has been suggested as a conformational sensor for the nucleotide state. To reprogram the Thermus thermophilus RNA helicase Hera for use of oxo-ATP instead of ATP and to investigate the sensor function of the Q-motif, we analyzed helicase activity of Hera Q28E. Crystal structures of the Hera N-terminal domain Q28E mutant (TthDEAD_Q28E) in apo- and ligand-bound forms show that Q28E does change specificity from adenine to 8-oxoadenine. However, significant structural changes accompany the Q28E mutation, which prevent the P-loop from adopting its catalytically active conformation and explain the lack of helicase activity of Hera_Q28E with either ATP or 8-oxo-ATP as energy sources. 8-Oxo-adenosine, 8-oxo-AMP, and 8-oxo-ADP weakly bind to TthDEAD_Q28E but in non-canonical modes. These results indicate that the Q-motif not only senses the nucleotide state of the helicase but could also stabilize a catalytically competent conformation of the P-loop and other helicase signature motifs.

Creatine supplementation normalizes mutagenesis of mitochondrial DNA as well as functional consequences.

Mutations of mitochondrial (mt) DNA play a role in neurodegeneration, normal aging, premature aging of the skin (photoaging), and tumors. We and others could demonstrate that mtDNA mutations can be induced in skin cells in vitro and in normal human skin in vivo by repetitive, sublethal ultraviolet (UV)-A-irradiation. These mutations are mediated by singlet oxygen and persist in human skin as long-term biomarkers of UV exposure. Although mtDNA exclusively encodes for the respiratory chain, involvement of the energy metabolism in mtDNA mutagenesis and a protective role of the energy precursor creatine have thus far not been shown. We assessed the amount of a marker mutation of mtDNA, the so-called common deletion, by real-time PCR. Induction of the common deletion was paralleled by a measurable decrease of oxygen consumption, mitochondrial membrane potential, and ATP content, as well as an increase of matrix metalloproteinase-1. Mitochondrial mutagenesis as well as functional consequences could be normalized by increasing intracellular creatine levels. These data indicate that increase of the energy precursor creatine protects from functionally relevant, aging-associated mutations of mitochondrial DNA.