Serine/arginine-rich splicing factors belong to a class of intrinsically disordered proteins

Serine/arginine-rich (SR) splicing factors play an important role in constitutive and alternative splicing as well as during several steps of RNA metabolism. Despite the wealth of functional information about SR proteins accumulated to-date, structural knowledge about the members of this family is very limited. To gain a better insight into structure-function relationships of SR proteins, we performed extensive sequence analysis of SR protein family members and combined it with ordered/disordered structure predictions. We found that SR proteins have properties characteristic of intrinsically disordered (ID) proteins. The amino acid composition and sequence complexity of SR proteins were very similar to those of the disordered protein regions. More detailed analysis showed that the SR proteins, and their RS domains in particular, are enriched in the disorder-promoting residues and are depleted in the order-promoting residues as compared to the entire human proteome. Moreover, disorder predictions indicated that RS domains of SR proteins were completely unstructured. Two different classification methods, the charge-hydropathy measure and the cumulative distribution function (CDF) of the disorder scores, were in agreement with each other, and they both strongly predicted members of the SR protein family to be disordered. This study emphasizes the importance of the disordered structure for several functions of SR proteins, such as for spliceosome assembly and for interaction with multiple partners. In addition, it demonstrates the usefulness of order/disorder predictions for inferring protein structure from sequence

Incorporation of genetic model parameters for cost-effective designs of genetic association studies using DNA pooling

<p>Abstract</p> <p>Background</p> <p>Studies of association methods using DNA pooling of single nucleotide polymorphisms (SNPs) have focused primarily on the effects of "machine-error", number of replicates, and the size of the pool. We use the non-centrality parameter (NCP) for the analysis of variance test to compute the approximate power for genetic association tests with DNA pooling data on cases and controls. We incorporate genetic model parameters into the computation of the NCP. Parameters involved in the power calculation are disease allele frequency, frequency of the marker SNP allele in coupling with the disease locus, disease prevalence, genotype relative risk, sample size, genetic model, number of pools, number of replicates of each pool, and the proportion of variance of the pooled frequency estimate due to machine variability. We compute power for different settings of number of replicates and total number of genotypings when the genetic model parameters are fixed. Several significance levels are considered, including stringent significance levels (due to the increasing popularity of 100 K and 500 K SNP "chip" data). We use a factorial design with two to four settings of each parameter and multiple regression analysis to assess which parameters most significantly affect power.</p> <p>Results</p> <p>The power can increase substantially as the genotyping number increases. For a fixed number of genotypings, the power is a function of the number of replicates of each pool such that there is a setting with maximum power. The four most significant parameters affecting power for association are: (1) genotype relative risk, (2) genetic model, (3) sample size, and (4) the interaction term between disease and SNP marker allele probabilities.</p> <p>Conclusion</p> <p>For a fixed number of genotypings, there is an optimal number of replicates of each pool that increases as the number of genotypings increases. Power is not substantially reduced when the number of replicates is close to but not equal to the optimal setting.</p

Turnover-Dependent Inactivation of the Nitrogenase MoFe-Protein at High pH

Proton uptake accompanies the reduction of all known substrates by nitrogenase. As a consequence, a higher pH should limit the availability of protons as a substrate essential for turnover, thereby increasing the proportion of more highly reduced forms of the enzyme for further study. The utility of the high-pH approach would appear to be problematic in view of the observation reported by Pham and Burgess [(1993) Biochemistry 32, 13725–13731] that the MoFe-protein undergoes irreversible protein denaturation above pH 8.65. In contrast, we found by both enzyme activity and crystallographic analyses that the MoFe-protein is stable when incubated at pH 9.5. We did observe, however, that at higher pHs and under turnover conditions, the MoFe-protein is slowly inactivated. While a normal, albeit low, level of substrate reduction occurs under these conditions, the MoFe-protein undergoes a complex transformation; initially, the enzyme is reversibly inhibited for substrate reduction at pH 9.5, yet in a second, slower process, the MoFe-protein becomes irreversibly inactivated as measured by substrate reduction activity at the optimal pH of 7.8. The final inactivated MoFe-protein has an increased hydrodynamic radius compared to that of the native MoFe-protein, yet it has a full complement of iron and molybdenum. Significantly, the modified MoFe-protein retains the ability to specifically interact with its nitrogenase partner, the Fe-protein, as judged by the support of ATP hydrolysis and by formation of a tight complex with the Fe-protein in the presence of ATP and aluminum fluoride. The turnover-dependent inactivation coupled to conformational change suggests a mechanism-based transformation that may provide a new probe of nitrogenase catalysis

Mechanism-Informed Refinement Reveals Altered Substrate-Binding Mode for Catalytically Competent Nitroreductase

Nitroreductase from Enterobacter cloacae (NR) reduces diverse nitroaromatics including herbicides, explosives and prodrugs, and holds promise for bioremediation, prodrug activation and enzyme-assisted synthesis. We solved crystal structures of NR complexes with bound substrate or analog for each of its two half-reactions. We complemented these with kinetic isotope effect (KIE) measurements elucidating H-transfer steps essential to each half-reaction. KIEs indicate hydride transfer from NADH to the flavin consistent with our structure of NR with the NADH analog nicotinic acid adenine dinucleotide (NAAD). The KIE on reduction of p-nitrobenzoic acid (p-NBA) also indicates hydride transfer, and requires revision of prior computational mechanisms. Our mechanistic information provided a structural restraint for the orientation of bound substrate, placing the nitro group closer to the flavin N5 in the pocket that binds the amide of NADH. KIEs show that solvent provides a proton, enabling accommodation of different nitro group placements, consistent with NR’s broad repertoire

Intrinsic Disorder Is a Common Feature of Hub Proteins from Four Eukaryotic Interactomes

Recent proteome-wide screening approaches have provided a wealth of information about interacting proteins in various organisms. To test for a potential association between protein connectivity and the amount of predicted structural disorder, the disorder propensities of proteins with various numbers of interacting partners from four eukaryotic organisms (Caenorhabditis elegans, Saccharomyces cerevisiae, Drosophila melanogaster, and Homo sapiens) were investigated. The results of PONDR VL-XT disorder analysis show that for all four studied organisms, hub proteins, defined here as those that interact with ≥10 partners, are significantly more disordered than end proteins, defined here as those that interact with just one partner. The proportion of predicted disordered residues, the average disorder score, and the number of predicted disordered regions of various lengths were higher overall in hubs than in ends. A binary classification of hubs and ends into ordered and disordered subclasses using the consensus prediction method showed a significant enrichment of wholly disordered proteins and a significant depletion of wholly ordered proteins in hubs relative to ends in worm, fly, and human. The functional annotation of yeast hubs and ends using GO categories and the correlation of these annotations with disorder predictions demonstrate that proteins with regulation, transcription, and development annotations are enriched in disorder, whereas proteins with catalytic activity, transport, and membrane localization annotations are depleted in disorder. The results of this study demonstrate that intrinsic structural disorder is a distinctive and common characteristic of eukaryotic hub proteins, and that disorder may serve as a determinant of protein interactivity

Conference Workshop Proceedings: Developing a Scholarship of Teaching and Learning Portfolio in Applied Horticulture

Preparing faculty to conduct quality teaching is critical to maximize student learning and the educational experience. As increased attention to faculty effectiveness and effect of their teaching program is observed, the more important it becomes for faculty to engage in the scholarship of teaching and learning (SoTL). The workshop “Developing a scholarship of teaching and learning portfolio in applied horticulture” was conducted at the 2022 American Society for Horticultural Science conference in Chicago, IL, USA, and featured a panel of teaching scholars who provided insight and guidance for developing, enhancing, evaluating, and promoting SoTL for both traditional classroom teachers and extension educators

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

LRTae: improving statistical power for genetic association with case/control data when phenotype and/or genotype misclassification errors are present

Abstract Background In the field of statistical genetics, phenotype and genotype misclassification errors can substantially reduce power to detect association with genetic case/control studies. Misclassification also can bias population frequency parameters such as genotype, haplotype, or multi-locus genotype frequencies. These problems are of particular concern in case/control designs because, short of repeated sampling, there is no way to detect misclassification errors. We developed a double-sampling procedure for case/control genetic association using a likelihood ratio test framework. Different approaches have been proposed to deal with misclassification errors. We have chosen the likelihood framework because of the ease with which misclassification probabilities may be incorporated into in the statistical framework and hypothesis testing. The statistic is called the Likelihood Ratio Test allowing for errors (LRTae) and is freely available via software download. Results We applied our procedure to 10,000 replicates of simulated case/control data in which we introduced phenotype misclassification errors. The phenotype considered is Ankylosing Spondylitis (AS). The LRTae method power was always greater than LRTstd power for the significance levels considered (5%, 1%, 0.1%, 0.01%). Power gains for the LRTae method over the LRTstd method increased as the significance level became more stringent. Multi-locus genotype frequency estimates using LRTae method were more accurate than estimates using LRTstd method. Conclusion The LRTae method can be applied to single-locus genotypes, multi-locus genotypes, or multi-locus haplotypes in a case/control framework and can be more powerful to detect association in case/control studies when both genotype and/or phenotype errors are present. Furthermore, the LRTae method provides asymptotically unbiased estimates of case and control genotype frequencies, as well as estimates of phenotype and/or genotype misclassification rates

INTERACTIVE APPLETS IN CALCULUS AND ENGINEERING COURSES

began work on a suite of computer applets (“MIT Mathlets”) designed to support basic MIT mathematics education. For a report on this work and a review of relevant literature see Miller and Upton [3]. The current state of this collection, along with supplementary material, can be found a