A Single DNA Point Mutation Leads to the Formation of a Cysteine-Tyrosine Crosslink in the Cysteine Dioxygenase from Bacillus subtilis.

Cysteine dioxygenase (CDO) is a non-heme iron-containing enzyme that catalyzes the oxidation of cysteine (Cys) to cysteine sulfinic acid (CSA). Crystal structures of eukaryotic CDOs revealed the presence of an unusual crosslink between the sulfur of a cysteine residue (C93 in Mus musculus CDO, MmCDO) and a carbon atom adjacent to the phenyl group of a tyrosine residue (Y157). Formation of this crosslink occurs over time as a byproduct of catalysis and increases the catalytic efficiency of CDO by at least 10-fold. Interestingly, in bacterial CDOs, the residue corresponding to C93 is replaced by a highly conserved glycine (G82 in Bacillus subtilis CDO, BsCDO), which precludes the formation of a C-Y crosslink in these enzymes; yet bacterial CDOs achieve turnover rates paralleling those of fully crosslinked eukaryotic CDOs. In the present study, we prepared the G82C variant of BsCDO to determine if a single DNA point mutation could lead to C-Y crosslink formation in this enzyme. We used gel electrophoresis, peptide mass spectrometry, electron paramagnetic resonance spectroscopy, and kinetic assays to characterize this variant alongside the natively crosslinked wild-type (WT) MmCDO and the natively non-crosslinked WT BsCDO. Collectively, our results provide compelling evidence that the G82C BsCDO variant is indeed capable of C-Y crosslink formation. Our kinetic studies indicate that G82C BsCDO has a reduced catalytic efficiency compared to WT BsCDO and that activity increases as the ratio of crosslinked to non-crosslinked enzyme increases. Finally, by carrying out a bioinformatic analysis of the CDO family, we were able to identify a large number of putatively crosslinked bacterial CDOs, the majority of which are from Gram-negative pathogenic bacteria.

Pathway of Hsp70 interactions at the ribosome.

In eukaryotes, an Hsp70 molecular chaperone triad assists folding of nascent chains emerging from the ribosome tunnel. In fungi, the triad consists of canonical Hsp70 Ssb, atypical Hsp70 Ssz1 and J-domain protein cochaperone Zuo1. Zuo1 binds the ribosome at the tunnel exit. Zuo1 also binds Ssz1, tethering it to the ribosome, while its J-domain stimulates Ssb's ATPase activity to drive efficient nascent chain interaction. But the function of Ssz1 and how Ssb engages at the ribosome are not well understood. Employing in vivo site-specific crosslinking, we found that Ssb(ATP) heterodimerizes with Ssz1. Ssb, in a manner consistent with the ADP conformation, also crosslinks to ribosomal proteins across the tunnel exit from Zuo1. These two modes of Hsp70 Ssb interaction at the ribosome suggest a functionally efficient interaction pathway: first, Ssb(ATP) with Ssz1, allowing optimal J-domain and nascent chain engagement; then, after ATP hydrolysis, Ssb(ADP) directly with the ribosome.

Covalent Modification of Amino Acids and Peptides Induced by Ionizing Radiation from an Electron Beam Linear Accelerator Used in Radiotherapy.

To identify modifications to amino acids that are directly induced by ionizing radiation, free amino acids and 3-residue peptides were irradiated using a linear accelerator (Linac) radiotherapy device. Mass spectrometry was performed to detail the relative sensitivity to radiation as well as identify covalent, radiation-dependent adducts. The order of reactivity of the 20 common amino acids was generally in agreement with published literature except for His (most reactive of the 20) and Cys (less reactive). Novel and previously identified modifications on the free amino acids were detected. Amino acids were far less reactive when flanked by glycine residues in a tripeptide. Order of reactivity, with GVG most and GEG least, was substantially altered, as were patterns of modification. Radiation reactivity of amino acids is clearly and strongly affected by conversion of the α-amino and α-carboxyl groups to peptide bonds, and the presence of neighboring amino acid residues.

In vivo cross-linking supports a head-to-tail mechanism for regulation of the plant plasma membrane P-type H+-ATPase.

In higher plants, a P-type proton-pumping ATPase generates the proton-motive force essential for the function of all other transporters and for proper growth and development. X-ray crystallographic studies of the plant plasma membrane proton pump have provided information on amino acids involved in ATP catalysis but provided no information on the structure of the C-terminal regulatory domain. Despite progress in elucidating enzymes involved in the signaling pathways that activate or inhibit this pump, the site of interaction of the C-terminal regulatory domain with the catalytic domains remains a mystery. Genetic studies have pointed to amino acids in various parts of the protein that may be involved, but direct chemical evidence for which ones are specifically interacting with the C terminus is lacking. In this study, we used in vivo cross-linking experiments with a photoreactive unnatural amino acid, p-benzoylphenylalanine, and tandem MS to obtain direct evidence that the C-terminal regulatory domain interacts with amino acids located within the N-terminal actuator domain. Our observations are consistent with a mechanism in which intermolecular, rather than intramolecular, interactions are involved. Our model invokes a "head-to-tail" organization of ATPase monomers in which the C-terminal domain of one ATPase molecule interacts with the actuator domain of another ATPase molecule. This model serves to explain why cross-linked peptides are found only in dimers and trimers, and it is consistent with prior studies suggesting that within the membrane the protein can be organized as homopolymers, including dimers, trimers, and hexamers.

A RecA protein surface required for activation of DNA polymerase V.

DNA polymerase V (pol V) of Escherichia coli is a translesion DNA polymerase responsible for most of the mutagenesis observed during the SOS response. Pol V is activated by transfer of a RecA subunit from the 3'-proximal end of a RecA nucleoprotein filament to form a functional complex called DNA polymerase V Mutasome (pol V Mut). We identify a RecA surface, defined by residues 112-117, that either directly interacts with or is in very close proximity to amino acid residues on two distinct surfaces of the UmuC subunit of pol V. One of these surfaces is uniquely prominent in the active pol V Mut. Several conformational states are populated in the inactive and active complexes of RecA with pol V. The RecA D112R and RecA D112R N113R double mutant proteins exhibit successively reduced capacity for pol V activation. The double mutant RecA is specifically defective in the ATP binding step of the activation pathway. Unlike the classic non-mutable RecA S117F (recA1730), the RecA D112R N113R variant exhibits no defect in filament formation on DNA and promotes all other RecA activities efficiently. An important pol V activation surface of RecA protein is thus centered in a region encompassing amino acid residues 112, 113, and 117, a surface exposed at the 3'-proximal end of a RecA filament. The same RecA surface is not utilized in the RecA activation of the homologous and highly mutagenic RumA'2B polymerase encoded by the integrating-conjugative element (ICE) R391, indicating a lack of structural conservation between the two systems. The RecA D112R N113R protein represents a new separation of function mutant, proficient in all RecA functions except SOS mutagenesis.

Disrupting KATP channels diminishes the estrogen-mediated protection in female mutant mice during ischemia-reperfusion.

BACKGROUND: Estrogen has been shown to mediate protection in female hearts against ischemia-reperfusion (I-R) stress. Composed by a Kir6.2 pore and an SUR2 regulatory subunit, cardiac ATP-sensitive potassium channels (KATP) remain quiescent under normal physiological conditions but they are activated by stress stimuli to confer protection to the heart. It remains unclear whether KATP is a regulatory target of estrogen in the female-specific I-R signaling pathway. In this study, we aimed at delineating the molecular mechanism underlying estrogen modulation on KATP channel activity during I-R. MATERIALS AND METHODS: We employed KATP knockout mice in which SUR2 is disrupted (SUR2KO) to characterize their I-R response using an in vivo occlusion model. To test the protective effects of estrogen, female mice were ovariectomized and implanted with 17ß-estradiol (E2) or placebo pellets (0.1 µg/g/day, 21-day release) before receiving an I-R treatment. Comparative proteomic analyses were performed to assess pathway-level alterations between KO-IR and WT-IR hearts. RESULTS AND DISCUSSION: Echocardiographic results indicated that KO females were pre-disposed to cardiac dysfunction at baseline. The mutant mice were more susceptible to I-R stress by having bigger infarcts (46%) than WT controls (31%). The observation was confirmed using ovariectomized mice implanted with E2 or placebo. However, the estrogen-mediated protection was diminished in KO hearts. Expression studies showed that the SUR2 protein level, but not RNA level, was up-regulated in WT-IR mice relative to untreated controls possibly via PTMs. Our antibodies detected different glycosylated SUR2 receptor species after the PNGase F treatment, suggesting that SUR2 could be modified by N-glycosylation. We subsequently showed that E2 could further induce the formation of complex-glycosylated SUR2. Additional time-point experiments revealed that I-R hearts had increased levels of N-glycosylated SUR2; and DPM1, the first committed step enzyme in the N-glycosylation pathway. Comparative proteomic profiling identified 41 differentially altered protein hits between KO-IR and WT-IR mice encompassing those related to estrogen biosynthesis. CONCLUSIONS: Our findings suggest that KATP is likely a downstream regulatory target of estrogen and it is indispensable in female I-R signaling. Increasing SUR2 expression by N-glycosylation mediated by estrogen may be effective to enhance KATP channel subunit expression in I-R.

Novel downstream molecular targets of SIRT1 in melanoma: a quantitative proteomics approach.

Melanoma is one of the most lethal forms of skin cancer and its incidence is continuing to rise in the United States. Therefore, novel mechanism and target-based strategies are needed for the management of this disease. SIRT1, a NAD(+)-dependent class III histone deacetylase, has been implicated in a variety of physiological processes and pathological conditions. We recently demonstrated that SIRT1 is upregulated in melanoma and its inhibition by a small-molecule, tenovin-1, inhibits cell proliferation and clonogenic survival of melanoma cells, possibly via activating p53. Here, we employed a gel free quantitative proteomics approach to identify the downstream effectors and targets of SIRT1 in melanoma. The human malignant melanoma, G361 cells were treated with tenovin-1 followed by protein extraction, in liquid trypsin digestion, and peptide analyses using nanoLC-MS/MS. A total of 1091 proteins were identified, of which 20 proteins showed significant differential expression with 95% confidence interval. These proteins were subjected to gene ontology and Ingenuity Pathway Analysis (IPA) to obtain the information regarding their biological and molecular functions. Real-Time qRT-PCR validation showed that five of these (PSAP, MYO1B, MOCOS, HIS1H4A and BUB3) were differentially expressed at mRNA levels. Based on their important role in cell cycle regulation, we selected to focus on BUB family proteins (BUB3, as well as BUB1 and BUBR1) for subsequent validation. The qRT-PCR and immunoblot analyses showed that tenovin-1 inhibition of SIRT1 resulted in a downregulation of BUB3, BUB1 and BUBR1 in multiple melanoma cell lines. Since tenovin-1 is an inhibitor of both SIRT1 and SIRT2, we employed lentivirus mediated silencing of SIRT1 and SIRT2 in G361 cells to determine if the observed effects on BUB family proteins are due to SIRT1- or SIRT2- inhibition. We found that only SIRT1 inhibition resulted in a decrease in BUB3, BUB1 and BUBR1. Our study identified the mitotic checkpoint regulator BUB family proteins as novel downstream targets of SIRT1. However, further validation is needed in appropriate models to confirm our findings and expand on our observations.

A peptide hormone and its receptor protein kinase regulate plant cell expansion.

Plant cells are immobile; thus, plant growth and development depend on cell expansion rather than cell migration. The molecular mechanism by which the plasma membrane initiates changes in the cell expansion rate remains elusive. We found that a secreted peptide, RALF (rapid alkalinization factor), suppresses cell elongation of the primary root by activating the cell surface receptor FERONIA in Arabidopsis thaliana. A direct peptide-receptor interaction is supported by specific binding of RALF to FERONIA and reduced binding and insensitivity to RALF-induced growth inhibition in feronia mutants. Phosphoproteome measurements demonstrate that the RALF-FERONIA interaction causes phosphorylation of plasma membrane H(+)-adenosine triphosphatase 2 at Ser(899), mediating the inhibition of proton transport. The results reveal a molecular mechanism for RALF-induced extracellular alkalinization and a signaling pathway that regulates cell expansion.

Genome, transcriptome, and secretome analysis of wood decay fungus Postia placenta supports unique mechanisms of lignocellulose conversion.

Brown-rot fungi such as Postia placenta are common inhabitants of forest ecosystems and are also largely responsible for the destructive decay of wooden structures. Rapid depolymerization of cellulose is a distinguishing feature of brown-rot, but the biochemical mechanisms and underlying genetics are poorly understood. Systematic examination of the P. placenta genome, transcriptome, and secretome revealed unique extracellular enzyme systems, including an unusual repertoire of extracellular glycoside hydrolases. Genes encoding exocellobiohydrolases and cellulose-binding domains, typical of cellulolytic microbes, are absent in this efficient cellulose-degrading fungus. When P. placenta was grown in medium containing cellulose as sole carbon source, transcripts corresponding to many hemicellulases and to a single putative beta-1-4 endoglucanase were expressed at high levels relative to glucose-grown cultures. These transcript profiles were confirmed by direct identification of peptides by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Also up-regulated during growth on cellulose medium were putative iron reductases, quinone reductase, and structurally divergent oxidases potentially involved in extracellular generation of Fe(II) and H(2)O(2). These observations are consistent with a biodegradative role for Fenton chemistry in which Fe(II) and H(2)O(2) react to form hydroxyl radicals, highly reactive oxidants capable of depolymerizing cellulose. The P. placenta genome resources provide unparalleled opportunities for investigating such unusual mechanisms of cellulose conversion. More broadly, the genome offers insight into the diversification of lignocellulose degrading mechanisms in fungi. Comparisons with the closely related white-rot fungus Phanerochaete chrysosporium support an evolutionary shift from white-rot to brown-rot during which the capacity for efficient depolymerization of lignin was lost.