Stress granules (SGs) are well characterized cytoplasmic RNA bodies that form under various stress conditions. We have observed that exposure of mammalian cells in culture to low doses of UVC induces the formation of discrete cytoplasmic RNA granules that were detected by immunofluorescence staining using antibodies to RNA-binding proteins. UVC-induced cytoplasmic granules are not Processing Bodies (P-bodies) and are bone fide SGs as they contain TIA-1, TIA-1/R, Caprin1, FMRP, G3BP1, PABP1, well known markers, and mRNA. Concomitant with the accumulation of the granules in the cytoplasm, cells enter a quiescent state, as they are arrested in G1 phase of the cell cycle in order to repair DNA damages induced by UVC irradiation. This blockage persists as long as the granules are present. A tight correlation between their decay and re-entry into S-phase was observed. However the kinetics of their formation, their low number per cell, their absence of fusion into larger granules, their persistence over 48 hours and their slow decay, all differ from classical SGs induced by arsenite or heat treatment. The induction of these SGs does not correlate with major translation inhibition nor with phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α). We propose that a restricted subset of mRNAs coding for proteins implicated in cell cycling are removed from the translational apparatus and are sequestered in a repressed form in SGs.

During the initial growth infection stage of Mycobacterium tuberculosis (Mtb), • NO produced by host macrophages inhibits heme-containing terminal cytochrome oxidases, inactivates iron/sulfur proteins and promotes entry into latency. Here we evaluate the potential of • NO as an inhibitor of Mtb cytochrome P450 enzymes, as represented by CYP130, CYP51 and the two previously uncharacterized enzymes CYP125 and CYP142. Using UV-visible absorption, resonance Raman, and stopped-flow spectroscopy, we investigated the reactions of • NO with these hemeproteins in their ferric resting form.• NO coordinates tightly to CYP125 and CYP142 (submicromolar), and with a lower affinity (micromolar) to CYP130 and CYP51. Anaerobic reduction of the ferric-NO species with sodium dithionite led to the formation of two spectrally distinct classes of five-coordinate ferrous-NO complexes. Exposure of these species to O 2 revealed that the ferrous-NO forms of CYP125 and CYP142 are labile and convert back to the ferric state within a few minutes, whereas ferrous CYP130 and CYP51 bind • NO almost irreversibly. This work clearly indicates that, at physiological concentrations (≈ 1 μM), • NO would impair the activity of CYP130 and CYP51, whereas CYP125 and CYP142 are more resistant. Selective P450 inhibition may contribute to the inhibitory effects of • NO on Mtb growth.Mycobacterium tuberculosis (Mtb) 1 continues to be an enormous threat to human health. Indeed, Mtb infects over 1.8 billion people worldwide and causes 1.5 million deaths per year. In immunocompetent individuals, the innate and adaptive arms of the immune system are relatively efficient in containing and killing Mtb. It is estimated that of 100 people newly infected with the tubercle bacilli, only about 5-10 individuals will develop tuberculosis over their lifetime (1). Host cells that are protective against Mtb include macrophages, dendritic cells, T-lymphocytes, and airway epithelial cells (2). The production of reactive oxygen intermediates and reactive nitrogen intermediates by innate immune cells is considered to be *To whom correspondence should be addressed. Telephone: (415) 476-2903. Fax: (415) 502-4728. E-mail: E-mail: ortiz@cgl.ucsf.edu.. ‡ University of California at San Francisco § Laval University SUPPORTING INFORMATION AVAILABLE The list of PCR primers for the cloning of cyp genes studied here are provided in Table S1. Spectral data from the reduction of ferric P450 enzymes by sodium dithionite in the absence or presence of CO are presented in Figure S1 and S2. The wavelength maxima of the calculated spectra and kinetic rates obtained from the reduction of the ferric and ferric-nitrosyl derivatives by sodium dithionite are listed in Tables S2 to S7. Selected representive real spectra from the reduction of the ferric-NO complexes by sodium dithionite are shown in Figure S3. Table S8 lists the wavelength maxima of the electronic absorption spectra of the ferric-and ferrous-nitrosyls complexes of selected thiolate-ligated hemeproteins. A list of relevant re...

Residues surrounding and interacting with the heme proximal ligand are important for efficient catalysis by heme proteins. The nitric oxide synthases (NOSs) are thiolate-coordinated enzymes that catalyze the hydroxylation of l-Arg in the first of the two catalytic cycles needed to synthesize nitric oxide. In NOSs, the indole NH group of a conserved tryptophan [W56 of the bacterial NOS-like protein from Staphylococcus aureus (saNOS)] forms a hydrogen bond with the heme proximal cysteinate ligand. The purpose of this study was to determine the impact of increasing (W56F and W56Y variants) or decreasing (W56H variant) the electron density of the proximal cysteinate ligand on molecular oxygen (O(2)) activation using saNOS as a model. We show that the removal of the indole NH···S(-) bond for W56F and W56Y caused an increase in the electron density of the cysteinate. This was probed by the decrease of the midpoint reduction potential (E(1/2)) along with weakened σ-bonding and strengthened π-backbonding with distal ligands (CO and O(2)). On the other hand, the W56H variant showed stronger Fe-OO and Fe-CO bonds (strengthened σ-bonding) along with an elevated E(1/2), which is consistent with the formation of a strong NH···S(-) hydrogen bond from H56. We also show here that changing the electron density of the proximal thiolate controls its "push effect"; whereas the rates of both O(2) activation and autoxidation of the Fe(II)O(2) complex increase with the stronger push effect created by removing the indole NH···S(-) hydrogen bond (W56F and W56Y variants), the W56H variant showed an increased stability of the complex against autoxidation and a slower rate of O(2) activation. These results are discussed with regard to the roles played by the conserved tryptophan-cysteinate interaction in the first catalytic cycle of NOS.

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