Novel enzymatic activity derived from the Semliki Forest virus capsid protein

The N-terminal segment of the Semliki Forest virus polyprotein is an intramolecular serine protease that cleaves itself off after the invariant Trp267 from a viral polyprotein and generates the mature capsid protein. After this autoproteolytic cleavage, the free carboxylic group of Trp267 interacts with the catalytic triad (His145, Asp167 and Ser219) and inactivates the enzyme. We have deleted the last 1-7 C-terminal residues of the mature capsid protease to investigate whether removal of Trp267 regenerates enzymatic activity. Although the C-terminally truncated polypeptides do not adopt a defined three-dimensional structure and show biophysical properties observed in natively unfolded proteins, they efficiently catalyse the hydrolysis of aromatic amino acid esters, with higher catalytic efficiency for tryptophan compared to tyrosine esters and k_cat/K_M values up to 5 × 10⁵ s⁻¹ M⁻¹. The enzymatic mechanism of these deletion variants is typical of serine proteases. The pH enzyme activity profile shows a pK_a1 = 6.9, and the Ser219Ala substitution destroys the enzymatic activity. In addition, the fast release of the first product of the enzymatic reaction is followed by a steady-state second phase, indicative of formation and breakdown of a covalent acyl-enzyme intermediate. The rates of acylation and deacylation are k₂ = 4.4±0.6 s⁻¹ and k₃ = 1.6±0.5 s⁻¹, respectively, for a tyrosine derivative ester substrate, and the amplitude of the burst phase indicates that 95% of the enzyme molecules are active. In summary, our data provide further evidence for the potential catalytic activity of natively unfolded proteins, and provide the basis for engineering of alphavirus capsid proteins towards hydrolytic enzymes with novel specificities.     

Mitochondrial leucine tRNA level and PTCD1 are regulated in response to leucine starvation

Pentatricopeptide repeat domain protein 1 (PTCD1) is a novel human protein that was recently shown to decrease the levels of mitochondrial leucine tRNAs. The physiological role of this regulation, however, remains unclear. Here we show that amino acid starvation by leucine deprivation significantly increased the mRNA steady-state levels of PTCD1 in human hepatocarcinoma (HepG2) cells. Amino acid starvation also increased the mitochondrially encoded leucine tRNA (tRNA^Leu(CUN)) and the mRNA for the mitochondrial leucyl-tRNA synthetase (LARS2). Despite increased PTCD1 mRNA steady-state levels, amino acid starvation decreased PTCD1 on the protein level. Decreasing PTCD1 protein concentration increases the stability of the mitochondrial leucine tRNAs, tRNA^Leu(CUN) and tRNA^Leu(UUR) as could be shown by RNAi experiments against PTCD1. Therefore, it is likely that decreased PTCD1 protein contributes to the increased tRNA^Leu(CUN) levels in amino acid-starved cells. The stabilisation of the mitochondrial leucine tRNAs and the upregulation of the mitochondrial leucyl-tRNA synthetase LARS2 might play a role in adaptation of mitochondria to amino acid starvation.     

A Conserved Region between the TPR and Activation Domains of p67phox Participates in Activation of the Phagocyte NADPH Oxidase

The phagocyte NADPH oxidase, dormant in resting cells, is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The membrane-integrated protein gp91^phox serves as the catalytic core, because it contains a complete electron-transporting apparatus from NADPH to molecular oxygen for superoxide production. Activation of gp91^phox requires the cytosolic proteins p67^phox, p47^phox, and Rac (a small GTPase). p67^phox, comprising 526 amino acids, moves upon cell stimulation to the membrane together with p47^phox and there interacts with Rac; these processes are prerequisite for gp91^phox activation. Here we show that a region of p67^phox (amino acids 190–200) C-terminal to the Rac-binding domain is evolutionarily well conserved and participates in oxidase activation at a later stage in conjunction with an activation domain. Alanine substitution for Tyr-198, Leu-199, or Val-204 abrogates the ability of p67^phox to support superoxide production by gp91^phox-based oxidase as well as its related oxidases Nox1 and Nox3; the activation also involves other invariant residues such as Leu-193, Asp-197, and Gly-200. Intriguingly, replacement of Gln-192 by alanine or that of Tyr-198 by phenylalanine or tryptophan rather enhances superoxide production by gp91^phox-based oxidase, suggesting a tuning role for these residues. Furthermore, the Y198A/V204A or L199A/V204A substitution leads to not only a complete loss of the activity of the reconstituted oxidase system but also a significant decrease in p67^phox interaction with the gp91^phox NADPH-binding domain, although these mutations affect neither the protein integrity nor the Rac binding activity. Thus the extended activation domain of p67^phox (amino acids 190–210) containing the D(Y/F)LGK motif plays an essential role in oxidase activation probably by interacting with gp91^phox.     

Overexpression of Eukaryotic Translation Elongation Factor 3 Impairs Gcn2 Protein Activation

In eukaryotes, phosphorylation of translation initiation factor 2α (eIF2α) by the kinase Gcn2 (general control nonderepressible 2) is a key response to amino acid starvation. Sensing starvation requires that Gcn2 directly contacts its effector protein Gcn1, and both must contact the ribosome. We have proposed that Gcn2 is activated by uncharged tRNA bound to the ribosomal decoding (A) site, in a manner facilitated by ribosome-bound Gcn1. Protein synthesis requires cyclical association of eukaryotic elongation factors (eEFs) with the ribosome. Gcn1 and Gcn2 are large proteins, raising the question of whether translation and monitoring amino acid availability can occur on the same ribosome. Part of the ribosome-binding domain in Gcn1 has homology to one of the ribosome-binding domains in eEF3, suggesting that these proteins utilize overlapping binding sites on the ribosome and consequently cannot function simultaneously on the same ribosome. Supporting this idea, we found that eEF3 overexpression in Saccharomyces cerevisiae diminished growth on amino acid starvation medium (Gcn⁻ phenotype) and decreased eIF2α phosphorylation, and that the growth defect associated with constitutively active Gcn2 was diminished by eEF3 overexpression. Overexpression of the eEF3 HEAT domain, or C terminus, was sufficient to confer a Gcn⁻ phenotype, and both fragments have ribosome affinity. eEF3 overexpression did not significantly affect Gcn1-ribosome association, but it exacerbated the Gcn⁻ phenotype of Gcn1-M7A that has reduced ribosome affinity. Together, this suggests that eEF3 blocks Gcn1 regulatory function on the ribosome. We propose that the Gcn1-Gcn2 complex only functions on ribosomes with A-site-bound uncharged tRNA, because eEF3 does not occupy these stalled complexes.     

Mutational analysis of potential pore-lining amino acids in TM IV of the Na/H exchanger

The Na⁺/H⁺ exchanger isoform 1 (NHE1) is an integral membrane protein that regulates intracellular pH by extruding an intracellular H⁺ in exchange for one extracellular Na⁺. In this study we examined the effect of site-specific mutagenesis on the pore-lining amino acid Phe161 and effects of mutagenesis on the charged amino acids Asp159 and Asp172. There was no absolute requirement for a carboxyl side chain at amino acid Asp159 or Asp172. Mutation of Asp159 to Asn or Gln maintained or increased the activity of the protein. Similarly, for Asp172, substitution with a Gln residue maintained activity of the protein, even though substitution with an Asn residue was inhibitory. The Asp172Glu mutant possessed normal activity after correction for its aberrant expression and surface targeting. Replacement of Phe161 with a Leu demonstrated that it was not irreplaceable in NHE1 function. However, the mutation Phe161lys inhibited NHE1 function, while the Phe161Ala mutation caused altered NHE1 targeting and expression levels. Our results show that these three amino acids, while being important in NHE1 function, are not irreplaceable. This study demonstrates that multiple substitutions at a single amino acid residue may be necessary to get a clearer picture membrane protein function.