The metastasis-associated protein S100A4 exists in several charged variants suggesting the presence of posttranslational modifications

<p>Abstract</p> <p>Background</p> <p>S100A4 is a metastasis-associated protein which has been linked to multiple cellular events, and has been identified extracellularly, in the cytoplasm and in the nucleus of tumor cells; however, the biological implications of subcellular location are unknown. Associations between a variety of posttranslational protein modifications and altered biological functions of proteins are becoming increasingly evident. Identification and characterization of posttranslationally modified S100A4 variants could thus contribute to elucidating the mechanisms for the many cellular functions that have been reported for this protein, and might eventually lead to the identification of novel drugable targets.</p> <p>Methods</p> <p>S100A4 was immuoprecipitated from a panel of <it>in vitro </it>and <it>in vivo </it>sources using a monoclonal antibody and the samples were separated by 2D-PAGE. Gels were analyzed by western blot and silver staining, and subsequently, several of the observed spots were identified as S100A4 by the use of MALDI-TOF and MALDI-TOF/TOF.</p> <p>Results</p> <p>A characteristic pattern of spots was observed when S100A4 was separated by 2D-PAGE suggesting the presence of at least three charge variants. These charge variants were verified as S100A4 both by western immunoblotting and mass spectrometry, and almost identical patterns were observed in samples from different tissues and subcellular compartments. Interestingly, recombinant S100A4 displayed a similar pattern on 2D-PAGE, but with different quantitative distribution between the observed spots.</p> <p>Conclusion</p> <p>Endogenously expressed S100A4 were shown to exist in several charge variants, which indicates the presence of posttranslational modifications altering the net charge of the protein. The different variants were present in all subcellular compartments and tissues/cell lines examined, suggesting that the described charge variants is a universal phenomenon, and cannot explain the localization of S100A4 in different subcellular compartments. However, the identity of the specific posttranslational modification and its potential contribution to the many reported biological events induced by S100A4, are subject to further studies.</p

Global, regional, and national incidence and mortality for HIV, tuberculosis, and malaria during 1990–2013: a systematic analysis for the Global Burden of Disease Study 2013

BACKGROUND: The Millennium Declaration in 2000 brought special global attention to HIV, tuberculosis, and malaria through the formulation of Millennium Development Goal (MDG) 6. The Global Burden of Disease 2013 study provides a consistent and comprehensive approach to disease estimation for between 1990 and 2013, and an opportunity to assess whether accelerated progress has occured since the Millennium Declaration. METHODS: To estimate incidence and mortality for HIV, we used the UNAIDS Spectrum model appropriately modified based on a systematic review of available studies of mortality with and without antiretroviral therapy (ART). For concentrated epidemics, we calibrated Spectrum models to fit vital registration data corrected for misclassification of HIV deaths. In generalised epidemics, we minimised a loss function to select epidemic curves most consistent with prevalence data and demographic data for all-cause mortality. We analysed counterfactual scenarios for HIV to assess years of life saved through prevention of mother-to-child transmission (PMTCT) and ART. For tuberculosis, we analysed vital registration and verbal autopsy data to estimate mortality using cause of death ensemble modelling. We analysed data for corrected case-notifications, expert opinions on the case-detection rate, prevalence surveys, and estimated cause-specific mortality using Bayesian meta-regression to generate consistent trends in all parameters. We analysed malaria mortality and incidence using an updated cause of death database, a systematic analysis of verbal autopsy validation studies for malaria, and recent studies (2010-13) of incidence, drug resistance, and coverage of insecticide-treated bednets. FINDINGS: Globally in 2013, there were 1·8 million new HIV infections (95% uncertainty interval 1·7 million to 2·1 million), 29·2 million prevalent HIV cases (28·1 to 31·7), and 1·3 million HIV deaths (1·3 to 1·5). At the peak of the epidemic in 2005, HIV caused 1·7 million deaths (1·6 million to 1·9 million). Concentrated epidemics in Latin America and eastern Europe are substantially smaller than previously estimated. Through interventions including PMTCT and ART, 19·1 million life-years (16·6 million to 21·5 million) have been saved, 70·3% (65·4 to 76·1) in developing countries. From 2000 to 2011, the ratio of development assistance for health for HIV to years of life saved through intervention was US$4498 in developing countries. Including in HIV-positive individuals, all-form tuberculosis incidence was 7·5 million (7·4 million to 7·7 million), prevalence was 11·9 million (11·6 million to 12·2 million), and number of deaths was 1·4 million (1·3 million to 1·5 million) in 2013. In the same year and in only individuals who were HIV-negative, all-form tuberculosis incidence was 7·1 million (6·9 million to 7·3 million), prevalence was 11·2 million (10·8 million to 11·6 million), and number of deaths was 1·3 million (1·2 million to 1·4 million). Annualised rates of change (ARC) for incidence, prevalence, and death became negative after 2000. Tuberculosis in HIV-negative individuals disproportionately occurs in men and boys (versus women and girls); 64·0% of cases (63·6 to 64·3) and 64·7% of deaths (60·8 to 70·3). Globally, malaria cases and deaths grew rapidly from 1990 reaching a peak of 232 million cases (143 million to 387 million) in 2003 and 1·2 million deaths (1·1 million to 1·4 million) in 2004. Since 2004, child deaths from malaria in sub-Saharan Africa have decreased by 31·5% (15·7 to 44·1). Outside of Africa, malaria mortality has been steadily decreasing since 1990. INTERPRETATION: Our estimates of the number of people living with HIV are 18·7% smaller than UNAIDS's estimates in 2012. The number of people living with malaria is larger than estimated by WHO. The number of people living with HIV, tuberculosis, or malaria have all decreased since 2000. At the global level, upward trends for malaria and HIV deaths have been reversed and declines in tuberculosis deaths have accelerated. 101 countries (74 of which are developing) still have increasing HIV incidence. Substantial progress since the Millennium Declaration is an encouraging sign of the effect of global action. FUNDING: Bill & Melinda Gates Foundation

Seeing Double: ASASSN-18bt Exhibits a Two-component Rise in the Early-time K2 Light

On 2018 February 4.41, the All-Sky Automated Survey for SuperNovae (ASAS-SN) discovered ASASSN-18bt in the K2 Campaign 16 field. With a redshift of z = 0.01098 and a peak apparent magnitude of B max = 14.31, ASASSN-18bt is the nearest and brightest SNe Ia yet observed by the Kepler spacecraft. Here we present the discovery of ASASSN-18bt, the K2 light curve, and prediscovery data from ASAS-SN and the Asteroid Terrestrial-impact Last Alert System. The K2 early-time light curve has an unprecedented 30-minute cadence and photometric precision for an SN Ia light curve, and it unambiguously shows a ~4 day nearly linear phase followed by a steeper rise. Thus, ASASSN-18bt joins a growing list of SNe Ia whose early light curves are not well described by a single power law. We show that a double-power-law model fits the data reasonably well, hinting that two physical processes must be responsible for the observed rise. However, we find that current models of the interaction with a nondegenerate companion predict an abrupt rise and cannot adequately explain the initial, slower linear phase. Instead, we find that existing published models with shallow 56Ni are able to span the observed behavior and, with tuning, may be able to reproduce the ASASSN-18bt light curve. Regardless, more theoretical work is needed to satisfactorily model this and other early-time SNe Ia light curves. Finally, we use Swift X-ray nondetections to constrain the presence of circumstellar material (CSM) at much larger distances and lower densities than possible with the optical light curve. For a constant-density CSM, these nondetections constrain ρ < 4.5 × 105 cm−3 at a radius of 4 × 1015 cm from the progenitor star. Assuming a wind-like environment, we place mass loss limits of for v w = 100 km s−1, ruling out some symbiotic progenitor systems. This work highlights the power of well-sampled early-time data and the need for immediate multiband, high-cadence follow-up for progress in understanding SNe Ia

An oxazetidine amino acid for chemical protein synthesis by rapid, serine-forming ligations

Amide-forming ligation reactions allow the chemical synthesis of proteins by the union of unprotected peptide segments, and enable the preparation of protein derivatives not accessible by expression or bioengineering approaches. The native chemical ligation (NCL) of thioesters and N-terminal cysteines is unquestionably the most successful approach, but is not ideal for all synthetic targets. Here we describe the synthesis of an Fmoc-protected oxazetidine amino acid for use in the α-ketoacid–hydroxylamine (KAHA) amide ligation. When incorporated at the N-terminus of a peptide segment, this four-membered cyclic hydroxylamine can be used for rapid serine-forming ligations with peptide α-ketoacids. This ligation operates at low concentration (100 μM–5 mM) and mild temperatures (20–25 °C). The utility of the reaction was demonstrated by the synthesis of S100A4, a 12 kDa calcium-binding protein not easily accessible by NCL or other amide-forming reactions due to its primary sequence and properties.ISSN:1755-4349ISSN:1755-433

Coupling S100A4 to Rhotekin alters Rho signaling output in breast cancer cells

Rho signaling is increasingly recognized to contribute to invasion and metastasis. In this study, we discovered that metastasis-associated protein S100A4 interacts with the Rho binding domain (RBD) of Rhotekin, thus connecting S100A4 to the Rho pathway. GST pull-down and immunoprecipitation assays demonstrated that S100A4 specifically and directly binds to Rhotekin RBD, but not other Rho effector RBDs. S100A4 binding to Rhotekin is calcium-dependent and uses residues distinct from those bound by active Rho. Interestingly, we found that S100A4 and Rhotekin can form a complex with active RhoA. Using RNAi, we determined that suppression of both S100A4 and Rhotekin leads to loss of Rho-dependent membrane ruffling in response to EGF, an increase in contractile F-actin “stress” fibers, and blocked invasive growth in three-dimensional culture. Accordingly, our data suggest that interaction of S100A4 and Rhotekin permits S100A4 to complex with RhoA and switch Rho function from stress fiber formation to membrane ruffling to confer an invasive phenotype