Browsing by Subject "Proteins"
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Item Ascites(1964-02-06) Combes, BurtonItem Cardiomyocyte Autophagy Is Induced by Protein Aggregation in Heart Disease(2009-06-19) Tannous, Paul; Hill, Joseph A.Autophagy is associated with diverse forms of myocardial stress. When I initiated my studies activators of this pathway had not been identified in the heart, nor was it clear weather autophagy is an adaptive or maladaptive response in the stressed myocardium. My initial research focused on autophagy in hypertension-induced heart failure, the most common cardiovascular disease in Western nations. Early evidence demonstrated generation of reactive oxygen species, protein damage, and protein aggregation in the acute period of pressure overload. Given the simultaneous presence of autophagosomes and aggregates, and autophagy's role in bulk degradation, I postulated these events were mechanistically linked. I designed experiments to test the hypotheses that protein aggregates are activators of autophagy in the heart, and that autophagy functions in aggregate clearance. Here I report novel findings that link pressure overload-induced protein aggregation to increased cardiomyocyte autophagy. Specifically, in the pressure-stressed ventricle 1) generation of reactive oxygen species is an early pathological event, 2) there is extensive protein aggregation with higher-order processing into aggresomes, 3) protein aggregation induces cardiomyocyte autophagy, and 4) in this setting autophagy functions in its role as a mechanism of bulk protein degradation. These findings are the first to demonstrate proteinopathy of non-genetic etiology contributes to hypertension-induced heart failure and that protein aggregates are robust activators of cardiomyocyte autophagy. To directly address the role of autophagy in cardiomyocyte clearance of toxic protein species, I turned my attention to CryABR120G-induced desmin-related cardiomyopathy (DRCM), an aggregate-associated disease with autosomal dominant inheritance. My studies demonstrated that 1) autophagy is activated by CryABR120G-induced protein aggregation, 2) aggregate formation is inversely proportional to the degree of autophagic activity and 3) blunting autophagy accelerates pathological myocardial remodeling and the onset of heart failure. Extending this work to clinical medicine, we observed increased autophagy in the skeletal muscle from patients with desmin-related skeletal myopathy. Cumulatively these data are the first to demonstrate autophagy is induced in DRCM and functions as a protective cellular response. These findings suggest autophagy is a pathway amenable to therapeutic intervention in patients suffering from myofibrillar myopathy, a disease class for which there are limited therapeutic options.Item Compositional Control of Phase-Separated Cellular Bodies(2018-04-04) Banani, Salman Ferozali; Yu, Hongtao; Thomas, Philip J.; Chen, Zhijian J.; Rosen, Michael K.Cellular bodies such as P bodies and PML nuclear bodies (PML NBs) appear to be phase separated liquids organized by multivalent interactions among proteins and RNA molecules. Although many components of various cellular bodies are known, general principles that define body composition are lacking. We have proposed a model for the formation of cellular bodies that is based on the polymerization-driven phase separation of key scaffold components of cellular bodies. We modeled cellular bodies using several engineered multivalent proteins and RNA. \textit{In vitro} and in cells these scaffold molecules form phase separated liquid droplets that are strongly enriched with the scaffold molecules. Analytical theories of polymerization suggest the resulting second phase contains large polymers of the multivalent scaffolds. Low valency client molecules partition differently into these structures depending on the stoichoimetric ratio of the scaffolds, with a sharp switch in recruitment across the phase diagram diagonal. Composition can switch rapidly through changes in scaffold concentration or valency. Natural PML NBs and P bodies show analogous partitioning behavior, suggesting how their compositions could be controlled by levels of PML SUMOylation or cellular mRNA concentration, respectively. Indeed, the engineered polySUMO/polySIM engineered scaffolds recruit many of the natural PML NB clients in a manner that depends on the SUMO:SIM stoichiometric ratio. Together, these data suggest a conceptual framework for considering the composition and control thereof of cellular bodies assembled through heterotypic multivalent interactions.Item Development and Application of Proteomic Technologies for the Analysis of Post-Translational Modifications(2007-08-08) Sprung, Robert William, Jr.; Zhao, YingmingPost-translational modifications represent a rapid and dynamic means for diversifying the chemistry of the ~20 ribosomally coded amino acids. As such, they provide an ideal mechanism for promoting cellular adaptability by facilitating the tuning of protein interactions and functions in response to changing environmental conditions. Despite their fundamental importance in regulating cellular functions and their wide implications in physiology, efficient means for the detection, enrichment and identification of proteins bearing specific modifications are lacking for most modifications. The availability of such methods would constitute invaluable tools supporting efforts to better understand the essential regulatory roles of modifications and the means by which aberrant modifications result in the onset and progression of disease. Towards this end, my dissertation describes the development and application of novel methods for the proteomic analysis of proteins bearing known modifications, including O-GlcNAc, lysine acetylation and methyl esterification. The identification of known targets of the modifications support some of the current ideas regarding their potential impact and serve as a means of validating the methods. More importantly, the identification of novel targets for the modifications challenges some currently held concepts, in particular regarding the relatively limited regulatory roles associated with lysine acetylation. In addition, the unparalleled power of proteomics as a screening strategy is demonstrated through compelling evidence of the existence of novel lysine acylations in vivo with respect to propionylation and butyrylation. Together, the methods described in this dissertation and the datasets generated embody powerful platforms and rich resources for the ongoing exploration of the fundamental contributions of post-translational modifications to the regulation of biological processes.Item Evolutionary Classification of Protein Domains: From Remote Homology to Family(2017-11-20) Liao, Yuxing; Rizo-Rey, José; Grishin, Nick V.; Rice, Luke M.; Tomchick, Diana R.Understanding the evolution of a protein, including both close and distant relationships, often reveals insight into its structure and function. A protein domain classification splits protein into domains and organizes them according to their evolutionary history. Existing classification databases fall back the speed of protein structure determination and do not include some known homologous relationships. I have participated in creating a hierarchical evolutionary classification of all proteins with experimentally determined spatial structures and developed a website for easy access and searches with keyword, sequence or structure (http://prodata.swmed.edu/ecod). ECOD (Evolutionary Classification Of protein Domains) is distinct from other structural classifications in that it groups domains primarily by evolutionary relationships (homology), rather than topology (or fold). Our database uniquely emphasizes distantly related homologs that are difficult to detect, and thus catalogs the largest number of evolutionary relationships among structural domain classifications. Placing distant homologs together underscores the ancestral similarities of these proteins and draws attention to the most important regions of sequence and structure, as well as conserved functional sites. The classification is assisted by an automated pipeline that classifies the most of new structures in Protein Data Bank weekly. This synchronization uniquely distinguishes ECOD among all protein classifications. For proteins that lack confident results from the automatic pipeline, I rely on information from literature, sequence and structure similarity scores, visual comparison and experience to classify them manually. I document the manual curation process in detail with an example of the remote homology between an autoproteolytic domain found in GPCR-Autoproteolysis Inducing domain, ZU5 and nucleoporin98. ECOD also recognizes closer relationships at the family level, initially with Pfam families. However, existing family databases do not cover all structures and disagree with ECOD in terms of domain definition and boundary. I generate multiple sequence alignment and profile for domains in the same family with structural information and demonstrate that the alignment quality is similar to manually checked Pfam seed alignments. I compare ECOD family profiles with Pfam and Conserved Domain Database and discuss about the improvement of domain boundary over known families and the dominance of small families in new families.Item Hierarchy of Interactions in Protein Evolution(2016-06-28) Salinas, Victor H.; Yu, Hongtao; Ranganathan, Rama; Takahashi, Joseph; Tu, BenjaminDeciphering the relationship between genotype and phenotype is complicated by the sheer number of possible cooperative interactions amongst the parts that make up biological systems. For even small systems such as individual protein domains, it has been difficult to comprehensively obtain high quality empirical data of amino acid interactions to distinguish different models for the global pattern of cooperativity. The statistical coupling analysis (SCA) - one approach for studying the co-evolution of amino acid positions in homologous sequences - provides a model for this pattern that is distinct from spatial proximity in tertiary structure, positional conservation, or even other forms of co-evolution. Here, we use an extension of deep mutational scanning to analyze nearly 50,000 single and double mutations in several homologs of a model protein - the PDZ family of protein interaction domains. Across the domains queried experimentally, the distributions of couplings between pairs of positions from all possible double mutants are well-approximated by unimodal distributions such that their average provides an estimate of the intrinsic coupling between them. Importantly, the SCA provides the best representation of this experimental pattern of couplings conserved among the homologs. These results highlight the heterogeneous pattern of couplings in protein structures and motivate the re-focus of efforts to understand protein folding and function toward the study of the origin of the co-evolving network of amino acids.Item Improving Profile Similarity Search and Alignment of Protein Sequences(2015-11-20) Tong, Jing; Ranganathan, Rama; Otwinowski, Zbyszek; Borek, Dominika; Grishin, Nick V.Protein function prediction is one of the most important problems in the field of computational biology. The most reliable method to predict protein function is to detect homologs. Homologous proteins tend to possess conserved sequence motifs, the same structure folds, and similar functional sites. Current sequence-based homology search methods are still unable to detect many similarities evident from protein spatial structures. We present a new method, COMPADRE, to assess the relationship between the query sequence and a hit in the database by considering the similarity between the query and hit's known homologs. This method markedly boosts the homology detection precision rate. Successful homology-based protein function prediction is also determined by accurate alignment between a protein sequence and its homolog. Alignment errors are the main bottleneck for homology modeling when the query is distantly related to the template. Alignment methods often misalign secondary structural elements by a few residues. We present a refinement method, SFESA, to improve pairwise sequence alignments by evaluating alignment variants generated by local shifts of template-defined secondary structures. The potential values of these methods for structure/function predictions are illustrated by the detection of homology between evolutionary distant yet structurally similar protein domains.Item Multi-Scale Structure and Dynamics of Visual Signaling in Drosophila Photoreceptor Cells(2012-07-16) Helms, Stephen Jess; Ranganathan, RamaA general problem in science today is how to understand complex systems. An emerging and promising approach makes the bold assumption that complex systems adhere to particular design principles. The power of this is that design principles by definition impose an intuitive nature on a system by presupposing purpose. Existing studies have fruitfully shown the application of engineering principles in biology, but biological systems have many distinct features, particularly due to evolution. In this work, I used Drosophila phototransduction, a well-studied sensory system renowned for its high performance, to search for evolutionary design principles. I focused on three levels of structure in the system: compartmentalization of molecules into microvilli, modularity of dynamic scaffolding by InaD, and functional integration within a single domain of InaD. Using rigorous quantitative measurement and theory with an evolutionary mindset, I uncovered intuitive, simplifying design principles at each level: Microvilli are used to build fast, homogeneous signaling compartments whose dimensions are constrained by these requirements. Dynamic scaffolding is a modular feature of InaD PDZs 4-5 which have been co-inherited in many scaffolds. Within PDZ5, ligand binding and oxidation of the domain are linked through pairwise coupling with a conformational equilibrium—a generic property found in all proteins—and not each other. These results show that this approach can be successful in revealing novel design principles in complex evolved systems.Item A New Approach to Optimize a Protein Energy Function on a Folding Pathway Using Gō-Like Potential and All-Atom, Ab Initio Monte Carlo Simulations(2016-01-19) Safronova, Aleksandra; Goldsmith, Elizabeth J.; Grishin, Nick V.; Otwinowski, Zbyszek; Rice, Luke M.Prediction of a protein structure is important for understanding the function of a protein. The process of protein structure prediction employs the approximation of a protein free energy that guides protein folding to the protein's native state. A function with a good approximation of the protein free energy should allow estimation of the structural distance of the evaluated candidate structure to the protein native state. Currently the energy optimization process relies on the correlation between the energy and the similarity to the native structure. The energy function is presented as a weighted sum of components which are designed by human experts with the use of statistical analysis of solved protein strictures. Values of the weights are derived through the procedure that maximizes the correlation between the energy and the similarity to the native structure measured by a root mean square deviation between coordinates of the protein backbone. Two major components are required for a successful ab initio modelling: (1) an effective energy function that discriminates the native protein structure out of all possible decoy structures; (2) an efficient sampling algorithm that quickly searches for the low-energy states. In this dissertation a new method for energy optimization is proposed. The method relies on a fast sampling algorithm and targets successful protein folding. The weights for energy components are optimized on a found with the Gō potential energy fast folding pathway. The Lennard-Jones potential, the Lazaridis-Karplus solvation potential, hydrogen bonding potential are used in the optimization algorithm. The optimized weights successfully predict all α and α/β proteins. The proposed strategy is conceptually different from the existing methods that optimize the energy on solved protein structures. The developed algorithm is a novel concept that allows the optimization of a more complex functional combination of the energy components that would improve the prediction quality.Item Optimization and Analysis of Weighted-Window Predictors of Structural Disorder in Proteins(2007-05-22) Holladay, Nathan Brent; Otwinowski, Zbyszek; Grishin, Nick V.; Pertsemlidis, Alexander; Rizo-Rey, JoséX-ray crystallographic protein structures often contain disordered regions that are observed as missing electron density. We have developed single sequence and profile-based weighted-window predictors of structural disorder in proteins, as well as a simple method for addressing disorder-prone chain termini in disorder prediction. Optimizing the parameters for these relatively simple predictors with crystallographic data using a simulated annealing type algorithm, we achieve performance similar to that of DISOPRED2. Optimized parameters from these disorder predictors provide information relating to physical processes underlying crystallographic disorder. Optimized score adjustment values suggest a simple, monotonic relationship between disorder and residue distance from termini that is nearly the same for amino- and carboxy-terminal positions. Residue disorder parameters are strongly associated with scales from certain experimental model systems that primarily reflect hydrophobic interactions. Our data do not suggest a strong association between crystallographic disorder and secondary structure beyond that explained by hydrophobicity. Our results lend support to the idea that while hydrophobic side chain interactions are primarily involved in determining stability of the folded conformation, hydrogen bonding and similar polar interactions are primarily involved in conformational and interaction specificity.Item Protein Mechanics Through X-Ray Crystallography(2016-04-14) White, Kristopher Ian; Rice, Luke M.; Ranganathan, Rama; Rosen, Michael K.; Yu, HongtaoProteins are dynamic entities which often cycle through a variety of conformational states as they carry out their functions. Despite the success of biophysical methods in determining the physical structures of proteins--that is, the precise three-dimensional configuration of all of their constituent atoms--no comprehensive physical models exist which accurately describe or predict the conformational cycling of proteins. For such models to be built, comprehensive knowledge of the energetics of intramolecular interactions in model proteins is essential. Here, we present three approaches which begin to address this problem, each through a different form of perturbation to a series of members of the PDZ protein family. First, we show that cycles of mutagenesis coupled with X-ray crystallography can reveal an anisotropic, distributed pattern of physical interactions in a PDZ domain, PSD-95 PDZ3. This pattern is functionally important and deeply connected to the evolution of PDZ domains in general. Second, we present a new approach for identifying essential dynamical features of proteins from relatively conventional X-ray diffraction data. Through combined analysis of nine different PDZ domain diffraction data sets, we show that collective features can be extracted and averaged, yielding a consensus picture of dynamics in the PDZ domain family. Finally, we report the development of a novel pump-probe method for directly inducing and reading out motions in proteins through the combined use of strong electric fields and time-resolved X-ray crystallography. We show that the method can be used to drive functionally relevant motions in a PDZ domain, LNX2 PDZ2, and provide a foundation for future efforts designed to directly probe the energetic architecture of proteins.Item [Southwestern News](2004-11-04) Siegfried, AmandaItem [Southwestern News](2002-09-20) Carter, WayneItem [Southwestern News](2001-07-05) Wren, Worth, Jr.Item [Southwestern News](2002-04-12) Shields, AmyItem [Southwestern News](2003-11-25) Shields, AmyItem [Southwestern News](2005-01-19) Siegfried, AmandaItem [Southwestern News](2001-06-29) Morrison, SusanItem [Southwestern News](1998-01-20) Steeves, Susan A.Item [Southwestern News](2004-11-25) Siem, Staishy Bostick
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