Molecular pathogenesis of human CD59 deficiency

Objective To characterize all 4 mutations described for CD59 congenital deficiency. Methods The 4 mutations, p.Cys64Tyr, p.Asp24Val, p.Asp24Valfs*, and p.Ala16Alafs*, were described in 13 individuals with CD59 malfunction. All 13 presented with recurrent Guillain-Barré syndrome or chronic inflammatory demyelinating polyneuropathy, recurrent strokes, and chronic hemolysis. Here, we track the molecular consequences of the 4 mutations and their effects on CD59 expression, localization, glycosylation, degradation, secretion, and function. Mutants were cloned and inserted into plasmids to analyze their expression, localization, and functionality. Results Immunolabeling of myc-tagged wild-type (WT) and mutant CD59 proteins revealed cell surface expression of p.Cys64Tyr and p.Asp24Val detected with the myc antibody, but no labeling by anti-CD59 antibodies. In contrast, frameshift mutants p.Asp24Valfs* and p.Ala16Alafs* were detected only intracellularly and did not reach the cell surface. Western blot analysis showed normal glycosylation but mutant-specific secretion patterns. All mutants significantly increased MAC-dependent cell lysis compared with WT. In contrast to CD59 knockout mice previously used to characterize phenotypic effects of CD59 perturbation, all 4 hCD59 mutations generate CD59 proteins that are expressed and may function intracellularly (4) or on the cell membrane (2). None of the 4 CD59 mutants are detected by known anti-CD59 antibodies, including the 2 variants present on the cell membrane. None of the 4 inhibits membrane attack complex (MAC) formation. Conclusions All 4 mutants generate nonfunctional CD59, 2 are expressed as cell surface proteins that may function in non–MAC-related interactions and 2 are expressed only intracellularly. Distinct secretion of soluble CD59 may have also a role in disease pathogenesis

Experimental library screening demonstrates the successful application of computational protein design to large structural ensembles

The stability, activity, and solubility of a protein sequence are determined by a delicate balance of molecular interactions in a variety of conformational states. Even so, most computational protein design methods model sequences in the context of a single native conformation. Simulations that model the native state as an ensemble have been mostly neglected due to the lack of sufficiently powerful optimization algorithms for multistate design. Here, we have applied our multistate design algorithm to study the potential utility of various forms of input structural data for design. To facilitate a more thorough analysis, we developed new methods for the design and high-throughput stability determination of combinatorial mutation libraries based on protein design calculations. The application of these methods to the core design of a small model system produced many variants with improved thermodynamic stability and showed that multistate design methods can be readily applied to large structural ensembles. We found that exhaustive screening of our designed libraries helped to clarify several sources of simulation error that would have otherwise been difficult to ascertain. Interestingly, the lack of correlation between our simulated and experimentally measured stability values shows clearly that a design procedure need not reproduce experimental data exactly to achieve success. This surprising result suggests potentially fruitful directions for the improvement of computational protein design technology

Increased versatility despite reduced molecular complexity evolution, structure and function of metazoan splicing factor PRPF39

In the yeast U1 snRNP the Prp39/Prp42 heterodimer is essential for early steps of spliceosome assembly. In metazoans no Prp42 ortholog exists, raising the question how the heterodimer is functionally substituted. Here we present the crystal structure of murine PRPF39, which forms a homodimer. Structure-guided point mutations disrupt dimer formation and inhibit splicing, manifesting the homodimer as functional unit. PRPF39 expression is controlled by NMD-inducing alternative splicing in mice and human, suggesting a role in adapting splicing efficiency to cell type specific requirements. A phylogenetic analysis reveals coevolution of shortened U1 snRNA and the absence of Prp42, which correlates with overall splicing complexity in different fungi. While current models correlate the diversity of spliceosomal proteins with splicing complexity, our study highlights a contrary case. We find that organisms with higher splicing complexity have substituted the Prp39/Prp42 heterodimer with a PRPF39 homodimer

Four small puzzles that Rosetta doesn't solve

A complete macromolecule modeling package must be able to solve the simplest structure prediction problems. Despite recent successes in high resolution structure modeling and design, the Rosetta software suite fares poorly on deceptively small protein and RNA puzzles, some as small as four residues. To illustrate these problems, this manuscript presents extensive Rosetta results for four well-defined test cases: the 20-residue mini-protein Trp cage, an even smaller disulfide-stabilized conotoxin, the reactive loop of a serine protease inhibitor, and a UUCG RNA tetraloop. In contrast to previous Rosetta studies, several lines of evidence indicate that conformational sampling is not the major bottleneck in modeling these small systems. Instead, approximations and omissions in the Rosetta all-atom energy function currently preclude discriminating experimentally observed conformations from de novo models at atomic resolution. These molecular "puzzles" should serve as useful model systems for developers wishing to make foundational improvements to this powerful modeling suite.Comment: Published in PLoS One as a manuscript for the RosettaCon 2010 Special Collectio

Mitigation of Urban Runoff Impacts on Atlanta Streams

Proceedings of the 1999 Georgia Water Resources Conference, March 30 and 31, Athens, Georgia.An interdisciplinary scientific panel was convened to assess the condition of Atlanta's streams and to identify those watershed management actions with the greatest potential to improve water quality and riparian and stream habitat in the Atlanta region. Broad recommendations included a description of elements to incorporate into a watershed management program for Atlanta, and specific suggestions for demonstration projects in four small, headwater watersheds. The four chosen demonstration sub-watersheds collectively reflect the gradient of impervious cover and stream quality present in Atlanta, and individually represent conditions comm.only observed throughout the area. Therefore, the general recommendations for these demonstration areas should be broadly applicable to the rest of the region.Sponsored and Organized by: U.S. Geological Survey, Georgia Department of Natural Resources, The University of Georgia, Georgia State University, Georgia Institute of TechnologyThis book was published by the Institute of Ecology, The University of Georgia, Athens, Georgia 30602-2202 with partial funding provided by the U.S. Department of Interior, geological Survey, through the Georgia Water Research Insttitute as authorized by the Water Research Institutes Authorization Act of 1990 (P.L. 101-397). The views and statements advanced in this publication are solely those of the authors and do not represent official views or policies of the University of Georgia or the U.S. Geological Survey or the conference sponsors

INFLUENCES OF WATERSHED URBANIZATION AND INSTREAM HABITAT ON MACROINVERTEBRATES IN COLD WATER STREAMS 1

We analyzed data from riffle and snag habitats for 39 small cold water streams with different levels of watershed urbanization in Wisconsin and Minnesota to evaluate the influences of urban land use and instream habitat on macroinvertebrate communities. Multivariate analysis indicated that stream temperature and amount of urban land use in the watersheds were the most influential factors determining macroinvertebrate assemblages. The amount of watershed urbanization was nonlinearly and negatively correlated with percentages of Ephemeroptera-Plecoptera-Trichoptera (EPT) abundance, EPT taxa, filterers, and scrapers and positively correlated with Hilsenhoff biotic index. High quality macroinvertebrate index values were possible if effective imperviousness was less than 7 percent of the watershed area. Beyond this level of imperviousness, index values tended to be consistently poor. Land uses in the riparian area were equal or more influential relative to land use elsewhere in the watershed, although riparian area consisted of only a small portion of the entire watershed area. Our study implies that it is extremely important to restrict watershed impervious land use and protect stream riparian areas for reducing human degradation on stream quality in low level urbanizing watersheds. Stream temperature may be one of the major factors through which human activities degrade cold-water streams, and management efforts that can maintain a natural thermal regime will help preserve stream quality.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72139/1/j.1752-1688.2003.tb03701.x.pd

Measurement of the CKM Matrix Element ∣Vcb∣|V_{cb}| from B0→D∗−ℓ+νℓB^{0} \to D^{*-} \ell^+ \nu_\ell at Belle

We present a new measurement of the CKM matrix element $|V_{cb}|$ from $B^{0} \to D^{*-} \ell^+ \nu_\ell$ decays, reconstructed with the full Belle data set of $711 \, \rm fb^{-1}$ integrated luminosity. Two form factor parameterizations, originally conceived by the Caprini-Lellouch-Neubert (CLN) and the Boyd, Grinstein and Lebed (BGL) groups, are used to extract the product $\mathcal{F}(1)\eta_{\rm EW}|V_{cb}|$ and the decay form factors, where $\mathcal{F}(1)$ is the normalization factor and $\eta_{\rm EW}$ is a small electroweak correction. In the CLN parameterization we find $\mathcal{F}(1)\eta_{\rm EW}|V_{cb}| = (35.06 \pm 0.15 \pm 0.56) \times 10^{-3}$, $\rho^{2}=1.106 \pm 0.031 \pm 0.007$, $R_{1}(1)=1.229 \pm 0.028 \pm 0.009$, $R_{2}(1)=0.852 \pm 0.021 \pm 0.006$. For the BGL parameterization we obtain $\mathcal{F}(1)\eta_{\rm EW}|V_{cb}|= (34.93 \pm 0.23 \pm 0.59)\times 10^{-3}$, which is consistent with the World Average when correcting for $\mathcal{F}(1)\eta_{\rm EW}$. The branching fraction of $B^{0} \to D^{*-} \ell^+ \nu_\ell$ is measured to be $\mathcal{B}(B^{0}\rightarrow D^{*-}\ell^{+}\nu_{\ell}) = (4.90 \pm 0.02 \pm 0.16)\%$. We also present a new test of lepton flavor universality violation in semileptonic $B$ decays, $\frac{{\cal B }(B^0 \to D^{*-} e^+ \nu)}{{\cal B }(B^0 \to D^{*-} \mu^+ \nu)} = 1.01 \pm 0.01 \pm 0.03~$. The errors correspond to the statistical and systematic uncertainties respectively. This is the most precise measurement of $\mathcal{F}(1)\eta_{\rm EW}|V_{cb}|$ and form factors to date and the first experimental study of the BGL form factor parameterization in an experimental measurement