Choreographing the DNA Damage Response: PP6 joins the dance

Comment on: Douglas P, et al. Protein phosphatase 6 interacts with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and dephosphorylates {gamma}-H2AX. Mol Cell Biol. 2010 Jan 11. [Epub ahead of print].     

Choreographing the fly's danse macabre

In several species, immune signaling networks are emerging as critical modulators of disease resistance, energy metabolism, and aging. In this issue of Cell Metabolism, Ren et al. (2007) lay the groundwork for dissecting the mechanisms of this coordination by characterizing the interplay between microbial pathogens and aging in the fly.     

STAP dance

Systematic image screening at EMBO Press uncovers many problems, most of which can be resolved. Standardized pre‐publication image screening would make for a more reliable literature. Flagging putative image manipulation post‐publication remains an important control mechanism, but must not fall victim to an overzealous response. The STAP stem cell papers are a case in point.     

On the enigma of old yellow enzyme's spectral properties

Old yellow enzyme (NADPH oxidoreductase) in the free and complexed state was thoroughly investigated by the following techniques: absorption, circular dichroism, fluorescence/phosphorescence and electron paramagnetic resonance spectroscopy and fluorescence/phosphorescence decay measurements, applied over a wide range of temperature (7-293K). The data obtained were interpreted by comparison with results from similar measurements on free FMN, existing spectral data on isoalloxazine model systems and theoretical data. The results clearly demonstrate the inadequacy of a simple phenolate-FMN donor-acceptor charge-transfer complex to explain the phenomena occurring upon the addition of phenols to old yellow enzyme. Instead it was found that the phenolate anion interferes strongly with an existing tight complex between FMN and the apoprotein, probably an H-bonded structure in which FMN is tautomerized and interacts with an L-chiral center. This is concluded from a separate electronic transition with an origin at 496 nm, thus far not recognized as such, and the circular dichroism observed. The emission is dominated by that of free FMN, although protein-bound FMN seems also to become luminescent in glassy solution at 143 K. A second fluorescence/phosphorescence emission appears upon excitation of both native and complexed old yellow enzyme in the ultraviolet. This emission is quenched by the addition of phenol to the enzyme, shows a large (3000-cm-1) blue shift on going to a low-temperature glass and is tentatively assigned to excimers of nucleic acids. Long-wavelength excitation with a synchronously pumped, mode-locked Rhodamine 6-G dye laser revealed a third, extremely weak emission in both native old yellow enzyme and its complexes. It decays with a lifetime of about 3 ns at 143 K. Electron paramagnetic resonance spectra revealed the presence of a low amount of an unpaired spin in old yellow enzyme. Owing to an unusual relaxational behaviour it could only be observed below 15 K and, again, the signal was measured in both the native enzyme and its complexes. Possible assignment and consequences of this observation are discussed. In frozen aqueous solutions of the enzyme-phenolate complex, a phase transition was discovered at which the colour of the complex reverted to that of the native enzyme. Subsequent melting restored the original colour. The observed phenomena and existing literature data lead to the conclusion that the only model from which no apparent inconsistencies emerge is that of a very complicated network of hydrogen-bonded structures in the protein. These involve several, partly unknown, chromophores. Phenols interfere with this network, leading to the formation of the long-wavelength absorption band in old yellow enzyme.     

Mec1 and Tel1: an arresting dance of resection

At sites of damage DNA is resected to enable the optimal form of repair, directed by homology. But how does the cell regulate resection while coordinating with the cell cycle? In this issue of The EMBO Journal, Clerici et al define missing links between DNA resection and cell cycle arrest during repair, showing that Mec1 can inhibit resection to subsequently activate arrest by Tel1.See also: M Clerici et al (December 2013)     

Amanitin-resistant RNA polymerase II mutations are in the enzyme's largest subunit

A fragment of the Drosophila melanogaster RpIIC4 locus, which encodes the RNA polymerase II subunit that determines amanitin sensitivity, was inserted into a bacterial plasmid cloning vehicle useful for over-production of hybrid proteins. Two plasmid constructions encoded hybrid proteins that reacted with antibodies against D. melanogaster RNA polymerase II. Use of subunit-specific antibodies indicated that these hybrid proteins displayed antigenic determinants unique to the largest polypeptide (215 kDa) of the enzyme. This RpII locus, the site at which mutations to amanitin-resistance occur, must therefore encode the largest polymerase II subunit.     

The ubiquitin signal and autophagy: an orchestrated dance leading to mitochondrial degradation

The quality of mitochondria, essential organelles that produce ATP and regulate numerous metabolic pathways, must be strictly monitored to maintain cell homeostasis. The loss of mitochondrial quality control systems is acknowledged as a determinant for many types of neurodegenerative diseases including Parkinson's disease (PD). The two gene products mutated in the autosomal recessive forms of familial early‐onset PD, Parkin and PINK1, have been identified as essential proteins in the clearance of damaged mitochondria via an autophagic pathway termed mitophagy. Recently, significant progress has been made in understanding how the mitochondrial serine/threonine kinase PINK1 and the E3 ligase Parkin work together through a novel stepwise cascade to identify and eliminate damaged mitochondria, a process that relies on the orchestrated crosstalk between ubiquitin/phosphorylation signaling and autophagy. In this review, we highlight our current understanding of the detailed molecular mechanisms governing Parkin‐/PINK1‐mediated mitophagy and the evidences connecting Parkin/PINK1 function and mitochondrial clearance in neurons.     

Conformational variability in an enzyme's active site: resonance Raman evidence for different acyl group conformations in N-acylglycine and N-acylalanine dithioacyl papains

It is demonstrated that the vibrational modes associated with the catalytically labile region of N-acylalanine dithioacyl papains undergo a major reorganization compared to the normal modes of corresponding model compounds. Thus, the resonance Raman (RR) spectrum of, e.g., N-benzoylalanine dithioacyl papain and its response to isotopic labeling cannot be understood completely on the basis of the RR spectrum of N-benzoylalanine ethyl dithio ester in one of its known conformational states [detailed in Lee, H., Angus, R. H., Storer, A. C., Varughese, K. I., & Carey, P. R. (1988) Biochemistry (preceding paper in this issue)]. This situation contrasts sharply to that for N-acylglycine dithioacyl papains whose RR spectra closely resemble those of the corresponding N-acylglycine ethyl dithio esters in a conformational state known as conformer B. For the N-acylalanine intermediates two possible causes are put forward to explain the rearrangement of the normal modes. First, the acyl groups based on alanine may bind in papain's active site in a conformation whose torsional angles near the -C(=S)S-group differ markedly from those of characterized model compounds. The second, and presently favored, explanation is that the N-acylalanine moiety is binding in the active site in an A- or C5-like conformation and that, in addition, there is significant vibrational coupling between some of the normal modes of the bound substrate and the normal modes associated with parts of the enzyme in contact with the substrate. The finding that deacylation for N-acylglycine or N-acylalanine dithioacyl papains must proceed from structures which are different is an indication that the mechanism of deacylation may not have strict stereochemical requirements.(ABSTRACT TRUNCATED AT 250 WORDS)