Paul J. Simpson

Uncoupling of GTP hydrolysis from eIF6 release on the ribosome causes Shwachman-Diamond syndrome

Removal of the assembly factor eukaryotic initiation factor 6 (eIF6) is critical for late cytoplasmic maturation of 60S ribosomal subunits. In mammalian cells, the current model posits that eIF6 release is triggered following phosphorylation of Ser 235 by activated protein kinase C. In contrast, genetic studies in yeast indicate a requirement for the ortholog of the SBDS (Shwachman-Bodian-Diamond syndrome) gene that is mutated in the inherited leukemia predisposition disorder Shwachman-Diamond syndrome (SDS). Here, by isolating late cytoplasmic 60S ribosomal subunits from Sbds-deleted mice, we show that SBDS and the GTPase elongation factor-like 1 (EFL1) directly catalyze eIF6 removal in mammalian cells by a mechanism that requires GTP binding and hydrolysis by EFL1 but not phosphorylation of eIF6 Ser 235. Functional analysis of disease-associated missense variants reveals that the essential role of SBDS is to tightly couple GTP hydrolysis by EFL1 on the ribosome to eIF6 release. Furthermore, complementary NMR spectroscopic studies suggest unanticipated mechanistic parallels between this late step in 60S maturation and aspects of bacterial ribosome disassembly. Our findings establish a direct role for SBDS and EFL1 in catalyzing the translational activation of ribosomes in all eukaryotes, and define SDS as a ribosomopathy caused by uncoupling GTP hydrolysis from eIF6 release.

Guidelines for Accurate and Transparent Health Estimates Reporting: the GATHER statement

Measurements of health indicators are rarely available for every population and period of interest, and available data may not be comparable. The Guidelines for Accurate and Transparent Health Estimates Reporting (GATHER) define best reporting practices for studies that calculate health estimates for multiple populations (in time or space) using multiple information sources. Health estimates that fall within the scope of GATHER include all quantitative population-level estimates (including global, regional, national, or subnational estimates) of health indicators, including indicators of health status, incidence and prevalence of diseases, injuries, and disability and functioning; and indicators of health determinants, including health behaviours and health exposures. GATHER comprises a checklist of 18 items that are essential for best reporting practice. A more detailed explanation and elaboration document, describing the interpretation and rationale of each reporting item along with examples of good reporting, is available on the GATHER website.

Characterization of two novel aldo-keto reductases from Arabidopsis: expression patterns, broad substrate specificity, and an open active-site structure suggest a role in toxicant metabolism following stress.

Aldo-keto reductases (AKRs) are widely distributed in nature and play numerous roles in the metabolism of steroids, sugars, and other carbonyls. They have also frequently been implicated in the metabolism of exogenous and endogenous toxicants, including those stimulated by stress. Although the Arabidopsis genome includes at least 21 genes with the AKR signature, very little is known of their functions. In this study, we have screened the Arabidopsis thaliana genomic sequence for genes with significant homology to members of the mammalian AKR1 family and identified four homologues for further study. Following alignment of the predicted protein sequences with representatives from the AKR superfamily, the proteins were ascribed not to the AKR1 family but to the AKR4C subfamily, with the individual designations of AKR4C8, AKR4C9, AKR4C10, and AKR4C11. Expression of two of the genes, AKR4C8 and AKR4C9, has been shown to be coordinately regulated and markedly induced by various forms of stress. The genes have been overexpressed in bacteria, and recombinant proteins have been purified and crystallized. Both enzymes display NADPH-dependent reduction of carbonyl compounds, typical of the superfamily, but will accept a very wide range of substrates, reducing a range of steroids, sugars, and aliphatic and aromatic aldehydes/ketones, although there are distinct differences between the two enzymes. We have obtained high-resolution crystal structures of AKR4C8 (1.4 A) and AKR4C9 (1.25 A) in ternary complexes with NADP(+) and acetate. Three extended loops, present in all AKRs and responsible for defining the cofactor- and substrate-binding sites, are shorter in the 4C subfamily compared to other AKRs. Consequently, the crystal structures reveal open and accommodative substrate-binding sites, which correlates with their broad substrate specificity. It is suggested that the primary role of these enzymes may be to detoxify a range of toxic aldehydes and ketones produced during stress, although the precise nature of the principal natural substrates remains to be determined.

AKR1C Isoforms Represent a Novel Cellular Target for Jasmonates alongside Their Mitochondrial-Mediated Effects

Members of the aldo-keto reductase (AKR) superfamily, particularly the AKR1C subfamily, are emerging as important mediators of the pathology of cancer. Agents that inhibit these enzymes may provide novel agents for either the chemoprevention or treatment of diverse malignancies. Recently, jasmonates, a family of plant stress hormones that bear a structural resemblance to prostaglandins, have been shown to elicit anticancer activities both in vitro and in vivo. In this study, we show that jasmonic acid (JA) and methyl jasmonate (MeJ) are capable of inhibiting all four human AKR1C isoforms. Although JA is the more potent inhibitor of recombinant AKR1C proteins, including the in vitro prostaglandin F synthase activity of AKR1C3, MeJ displayed greater potency in cellular systems that was, at least in part, due to increased cellular uptake of MeJ. Moreover, using the acute myelogenous leukemia cell lines HL-60 and KG1a, we found that although both jasmonates were able to induce high levels of reactive oxygen species in a dose-dependent fashion, only MeJ was able to induce high levels of mitochondrial superoxide (MSO), possibly as an epiphenomenon of mitochondrial damage. There was a strong correlation observed between MSO formation at 24 hours and reduced cellularity at day 5. In conclusion, we have identified AKR1C isoforms as a novel target of jasmonates in cancer cells and provide further evidence of the promise of these compounds, or derivatives thereof, as adjunctive therapies in the treatment of cancer.

CTNNBL1 Is a Novel Nuclear Localization Sequence-binding Protein That Recognizes RNA-splicing Factors CDC5L and Prp31

Nuclear proteins typically contain short stretches of basic amino acids (nuclear localization sequences; NLSs) that bind karyopherin α family members, directing nuclear import. Here, we identify CTNNBL1 (catenin-β-like 1), an armadillo motif-containing nuclear protein that exhibits no detectable primary sequence homology to karyopherin α, as a novel, selective NLS-binding protein. CTNNBL1 (a single-copy gene conserved from fission yeast to man) was previously found associated with Prp19-containing RNA-splicing complexes as well as with the antibody-diversifying enzyme AID. We find that CTNNBL1 association with the Prp19 complex is mediated by recognition of the NLS of the CDC5L component of the complex and show that CTNNBL1 also interacts with Prp31 (another U4/U6.U5 tri-snRNP-associated splicing factor) through its NLS. As with karyopherin αs, CTNNBL1 binds NLSs via its armadillo (ARM) domain, but displays a separate, more selective NLS binding specificity. Furthermore, the CTNNBL1/AID interaction depends on amino acids forming the AID conformational NLS with CTNNBL1-deficient cells showing a partial defect in AID nuclear accumulation. However, in further contrast to karyopherin αs, the CTNNBL1 N-terminal region itself binds karyopherin αs (rather than karyopherin β), suggesting a function divergent from canonical nuclear transport. Thus, CTNNBL1 is a novel NLS-binding protein, distinct from karyopherin αs, with the results suggesting a possible role in the selective intranuclear targeting or interactions of some splicing-associated complexes.

Experimental arthritis: Oral aggrecanase inhibitor may slow postinjury cartilage breakdown

AGG-523, an aggrecanase inhibitor, reduces synovial fluid levels of Ala–Arg– Gly-aggrecan (a major cartilage matrix breakdown product) in a rat model of knee injury, new findings show. “Our study suggests the potential of postinjury aggrecanase inhibition to stop or slow down immediate cartilage degradation as well as development of osteoarthritis years after injury in humans,” explains lead investigator, Priya Chockalingam. The researchers developed a highly sensitive and specific enzyme-linked immunosorbent assay, using commercially available reagents, which revealed elevated levels of Ala–Arg–Gly-aggrecan in synovial fluid from animals and humans with knee injury. Levels of this fragment in human knee synovial fluid increased by an average of 13.6-fold in the 12 weeks after cruciate ligament rupture and 8.1-fold after meniscal injury. Although Ala–Arg–Glyaggrecan levels then declined, they were still EXPERIMENTAL ARTHRITIS

Cardiac resuscitation: Assisted CPR improves outcomes

manual cardiopulmonary resuscitation (CPr) is inherently inefficient. alternative techniques are compression– decompression CPr, which improves forward blood flow on compression and returns the chest to the starting position, and applying negative intrathoracic pressure during decompression using impedance-threshold devices, which improve venous return to the heart. a new report shows that combining these two approaches improves 1-year survival—by nearly 50% versus manual CPr—for patients who have out-of-hospital cardiac arrests. the trial was conducted in 46 emergency medical service agencies in the usa. of 1,653 adults who had an out-of-hospital cardiac arrest and were enrolled in the study, 813 were randomly assigned to manual CPr and 840 to assisted CPr using the two approaches. of the patients who received manual CPr, 47 (6%) were discharged from hospital with good neurological function (modified rankin score ≤3), compared cardiac resuscitation