Ubiquitin Ser65 phosphorylation affects ubiquitin structure,chain assembly and hydrolysis

The protein kinase PINK1 was recently shown to phosphorylate ubiquitin (Ub) on Ser65, and phosphoUb activates the E3 ligase Parkin allosterically. Here, we show that PINK1 can phosphorylate every Ub in Ub chains. Moreover, Ser65 phosphorylation alters Ub structure, generating two conformations in solution. A crystal structure of the major conformation resembles Ub but has altered surface properties. NMR reveals a second phosphoUb conformation in which β5-strand slippage retracts the C-terminal tail by two residues into the Ub core. We further show that phosphoUb has no effect on E1-mediated E2 charging but can affect discharging of E2 enzymes to form polyUb chains. Notably, UBE2R1- (CDC34), UBE2N/UBE2V1- (UBC13/UEV1A), TRAF6- and HOIP-mediated chain assembly is inhibited by phosphoUb. While Lys63-linked poly-phosphoUb is recognized by the TAB2 NZF Ub binding domain (UBD), 10 out of 12 deubiquitinases (DUBs), including USP8, USP15 and USP30, are impaired in hydrolyzing phosphoUb chains. Hence, Ub phosphorylation has repercussions for ubiquitination and deubiquitination cascades beyond Parkin activation and may provide an independent layer of regulation in the Ub system.     

An AlphaScreen®-based assay for high-throughput screening for specific inhibitors of nuclear import

Specific viral proteins enter the nucleus of infected cells to perform essential functions, as part of the viral life cycle. The integrase (IN) molecule of human immunodeficiency virus (HIV)-1 is of particular interest in this context due to its integral role in integrating the HIV genome into that of the infected host cell. Most IN-based antiviral compounds target the IN/DNA interaction, but since IN must first enter the nucleus before it can perform these critical functions, nuclear transport of IN is also an attractive target for therapeutic intervention. Here the authors describe a novel high-throughput screening assay for identifying inhibitors of nuclear import, particularly IN, based on amplified luminescent proximity homogeneous assay (AlphaScreen(?)) technology, which is high throughput, requires low amounts of material, and is efficient and cost-effective. The authors use the assay to screen for specific inhibitors of the interaction between IN and its nuclear transport receptor importin α/β, successfully identifying several inhibitors of the IN/importin α/β interaction. Importantly, they demonstrate that one of the identified compounds, mifepristone, is effective in preventing active nuclear transport of IN in transfected cells and hence may represent a useful anti-HIV therapeutic. The screen also identified broad-spectrum importin α/β inhibitors such as ivermectin, which may represent useful tools for nuclear transport research in the future. The authors validate the activity and specificity of mifepristone and ivermectin in inhibiting nuclear protein import in living cells, underlining the utility of the screening approach.     

Cell wars: regulation of cell survival and proliferation by cell competition

During cell competition fitter cells take over the tissue at the expense of viable, but less fit, cells, which are eliminated by induction of apoptosis or senescence. This probably acts as a quality-control mechanism to eliminate suboptimal cells and safeguard organ function. Several experimental conditions have been shown to trigger cell competition, including differential levels in ribosomal activity or in signalling pathway activation between cells, although it is unclear how those differences are sensed and translated into fitness levels. Many of the pathways implicated in cell competition have been previously linked with cancer, and this has led to the hypothesis that cell competition could play a role in tumour formation. Cell competition could be co-opted by cancer cells to kill surrounding normal cells and boost their own tissue colonization. However, in some cases, cell competition could have a tumour suppressor role, as cells harbouring mutations in a subset of tumour suppressor genes are killed by wild-type cells. Originally described in developing epithelia, competitive interactions have also been observed in some stem cell niches, where they play a role in regulating stem cell selection, maintenance and tissue repopulation. Thus competitive interactions could be relevant to the maintenance of tissue fitness and have a protective role against aging.     

Importins and Beyond: Non-Conventional Nuclear Transport Mechanisms

The movement of proteins between the cytoplasm and the nucleus conventionally involves the recognition of nuclear targeting signals by members of the importin (Imp) superfamily of nuclear transporters, followed by translocation through the nuclear envelope-embedded nuclear pore complexes (NPCs). It is becoming increasingly apparent, however, that distinct alternative pathways for nuclear transport exist and are relatively abundant. This review examines several of these novel pathways, including facilitation of Imp-dependent transport by microtubule motors, and Imp-independent pathways involving either other transport molecules such as the calcium-binding protein calmodulin or through direct binding to the components of the NPC. The existence of these pathways and the fact that many proteins appear to possess separate Imp-dependent and -independent nuclear import mechanisms ensure that the cell can function under conditions in which Imp-dependent transport is inhibited and/or modulate the efficiency of Imp-dependent transport itself, according to the need.     

Snake Envenoming: A Disease of Poverty

Background

Most epidemiological and clinical reports on snake envenoming focus on a single country and describe rural communities as being at greatest risk. Reports linking snakebite vulnerability to socioeconomic status are usually limited to anecdotal statements. The few reports with a global perspective have identified the tropical regions of Asia and Africa as suffering the highest levels of snakebite-induced mortality. Our analysis examined the association between globally available data on snakebite-induced mortality and socioeconomic indicators of poverty.

Methodology/Principal Findings

We acquired data on (i) the Human Development Index, (ii) the Per Capita Government Expenditure on Health, (iii) the Percentage Labour Force in Agriculture and (iv) Gross Domestic Product Per Capita from publicly available databases on the 138 countries for which snakebite-induced mortality rates have recently been estimated. The socioeconomic datasets were then plotted against the snakebite-induced mortality estimates (where both datasets were available) and the relationship determined. Each analysis illustrated a strong association between snakebite-induced mortality and poverty.

Conclusions/Significance

This study, the first of its kind, unequivocally demonstrates that snake envenoming is a disease of the poor. The negative association between snakebite deaths and government expenditure on health confirms that the burden of mortality is highest in those countries least able to deal with the considerable financial cost of snakebite.     

Pre-clinical assays predict pan-African Echis viper efficacy for a species-specific antivenom

Background

Snakebite is a significant cause of death and disability in subsistent farming populations of sub-Saharan Africa. Antivenom is the most effective treatment of envenoming and is manufactured from IgG of venom-immunised horses/sheep but, because of complex fiscal reasons, there is a paucity of antivenom in sub-Saharan Africa. To address the plight of thousands of snakebite victims in savannah Nigeria, the EchiTAb Study Group organised the production, testing and delivery of antivenoms designed to treat envenoming by the most medically-important snakes in the region. The Echis saw-scaled vipers have a wide African distribution and medical importance. In an effort to maximise the clinical utility of scarce antivenom resources in Africa, we aimed to ascertain, at the pre-clinical level, to what extent the E. ocellatus-specific EchiTAbG antivenom, which was designed specifically for Nigeria, neutralised the lethal activity of venom from two other African species, E. pyramidum leakeyi and E. coloratus.

Methodology/Principal Findings

Despite apparently quite distinctive venom protein profiles, we observed extensive cross-species similarity in the immuno-reactivity profiles of Echis species-specific antisera. Using WHO standard pre-clinical in vivo tests, we determined that the monospecific EchiTAbG antivenom was as effective at neutralising the venom-induced lethal effects of E. pyramidum leakeyi and E. coloratus as it was against E. ocellatus venom. Under the restricted conditions of this assay, the antivenom was ineffective against the lethal effects of venom from the non-African Echis species, E. carinatus sochureki.

Conclusions/Significance

Using WHO-recommended pre-clinical tests we have demonstrated that the new anti-E. ocellatus monospecific antivenom EchiTAbG, developed in response to the considerable snakebite-induced mortality and morbidity in Nigeria, neutralised the lethal effects of venoms from Echis species representing each taxonomic group of this genus in Africa. This suggests that this monospecific antivenom has potential to treat envenoming by most, perhaps all, African Echis species.     

Phenotypic variability in X-linked ocular albinism: relationship to linkage genotypes.

One hundred nineteen individuals from 11 families with X-linked ocular albinism (OA1) were studied with respect to both their clinical phenotypes and their linkage genotypes. In a four-generation Australian family, two affected males and an obligatory carrier lacked cutaneous melanin macroglobules (MMGs); ocular features were identical to those of Nettleship-Falls OA1. Four other families had more unusual phenotypic features in addition to OA1. All OA1 families were genotyped at DXS16, DXS85, DXS143, STS, and DXS452 and for a CA-repeat polymorphism at the Kallmann syndrome locus (KAL). Separate two-point linkage analyses were performed for the following: group A, six families with biopsy-proved MMGs in at least one affected male; group B, four families whose biopsy status was not known; and group C, OA-9 only (16 samples), the family without MMGs. At the set of loci closest to OA1, there is no clear evidence in our data set for locus heterogeneity between groups A and C or among the four other families with complex phenotypes. Combined multipoint analysis (LINKMAP) in the 11 families and analysis of individual recombination events confirms that the major locus for OA1 resides within the DXS85-DXS143 interval. We suggest that more detailed clinical evaluations of OA1 individuals and families should be performed for future correlation with specific mutations in candidate OA1 genes.     

Detection of two-component mixtures of lognormal distributions in grouped, doubly truncated data: analysis of red blood cell volume distributions

We have examined the statistical requirements for the detection of mixtures of two lognormal distributions in doubly truncated data when the sample size is large. The expectation-maximization algorithm was used for parameter estimation. A bootstrap approach was used to test for a mixture of distributions using the likelihood ratio statistic. Analysis of computer simulated mixtures showed that as the ratio of the difference between the means to the minimum standard deviation increases, the power for detection also increases and the accuracy of parameter estimates improves. These procedures were used to examine the distribution of red blood cell volume in blood samples. Each distribution was doubly truncated to eliminate artifactual frequency counts and tested for best fit to a single lognormal distribution or a mixture of two lognormal distributions. A single population was found in samples obtained from 60 healthy individuals. Two subpopulations of cells were detected in 25 of 27 mixtures of blood prepared in vitro. Analyses of mixtures of blood from 40 patients treated for iron-deficiency anemia showed that subpopulations could be detected in all by 6 weeks after onset of treatment. To determine if two-component mixtures could be detected, distributions were examined from untransfused patients with refractory anemia. In two patients with inherited sideroblastic anemia a mixture of microcytic and normocytic cells was found, while in the third patient a single population of microcytic cells was identified. In two family members previously identified as carriers of inherited sideroblastic anemia, mixtures of microcytic and normocytic subpopulations were found. Twenty-five patients with acquired myelodysplastic anemia were examined. A good fit to a mixture of subpopulations containing abnormal microcytic or macrocytic cells was found in two. We have demonstrated that with large sample sizes, mixtures of distributions can be detected even when distributions appear to be unimodal. These statistical techniques provide a means to characterize and quantify alterations in erythrocyte subpopulations in anemia but could also be applied to any set of grouped, doubly truncated data to test for the presence of a mixture of two lognormal distributions.     

OA1 mutations and deletions in X-linked ocular albinism.

X-linked ocular albinism (OA1), Nettleship-Falls type, is characterized by decreased ocular pigmentation, foveal hypoplasia, nystagmus, photodysphoria, and reduced visual acuity. Affected males usually demonstrate melanin macroglobules on skin biopsy. We now report results of deletion and mutation screening of the full-length OA1 gene in 29 unrelated North American and Australian X-linked ocular albinism (OA) probands, including five with additional, nonocular phenotypic abnormalities (Schnur et al. 1994). We detected 13 intragenic gene deletions, including 3 of exon 1, 2 of exon 2, 2 of exon 4, and 6 others, which span exons 2-8. Eight new missense mutations were identified, which cluster within exons 1, 2, 3, and 6 in conserved and/or putative transmembrane domains of the protein. There was also a splice acceptor-site mutation, a nonsense mutation, a single base deletion, and a previously reported 17-bp exon 1 deletion. All patients with nonocular phenotypic abnormalities had detectable mutations. In summary, 26 (approximately 90%) of 29 probands had detectable alterations of OA1, thus confirming that OA1 is the major locus for X-linked OA.