Browsing by Subject "Thioredoxins"
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Item Algorithmic Developments for Sequence Analysis, Structure Modeling and Functional Prediction of Proteins(2006-12-20) Qi, Yuan; Grishin, Nick V.Sequence, structure and function, being the three most important properties of proteins, are interrelated through homology relationships. In this post-genome era, we are equipped with abundant sequence information. Homology inference is thus of great practical importance because of its ability to make structural and functional predictions through sequence analysis. In an effort to explore and utilize the protein sequence-structure-function relationships, with homology detection and utilization as the central scheme, this work concentrates on algorithmic development of methods and systems for sequence similarity search, structure modeling and functional prediction purposes, as well as performs structure prediction and classification for specific protein families. Three algorithmic developments are described in this dissertation. First, to facilitate identification of structurally or functionally important interactions between positions in a protein family, a program has been developed to perform positional correlation analysis of multiple sequence alignments using different methods. The program has been shown to be useful to identify functionally important position pairs or networks of correlated positions. Second, to further increase the sensitivity of sequence similarity search methods in terms of homology detection and structure modeling ability, a method has been developed by incorporating predicted secondary structure information with sequence profiles. Evaluation on PFAM-based system shows that this method provides improved structure template detection ability and generates alignment of better quality. Third, in order to systematically assess the structure modeling abilities of different sequence similarity search programs, a comprehensive evaluation system has been developed. This large-scale automatic evaluation system assesses the fold recognition ability and alignment quality of different programs from global and local perspectives using both reference-dependent and reference-independent approaches, which provides an instrument to understand the progress and limitations of the field. Two structure prediction and classification projects using manual analysis and existing tools are also described in this dissertation. First, the structure of C-terminal domain of Gyrase A is predicted through inferred homology relationship with regulator of chromosome condensation (RCC1). This prediction has been validated by experimental data. Second, a hierarchical structure classification of thioredoxin-like fold proteins has been carried out, which promotes understanding of fold definitions and sequence-structure-function relationshipsItem Thioredoxin-1 Inhibition Promotes MLKL Activation: Biochemical Insights Into a Suppressor of the Necroptotic Pathway(2017-07-13) Reynoso, Eduardo; Amatruda, James F.; Wang, Zhigao; Abrams, John M.; Chen, Zhijian J.Necroptosis is an immunogenic caspase-independent cell death program. While mainly serving to defend against viral infection, dysregulated necroptotic signaling contributes to the pathology of a growing number of human diseases associated with inflammation, neurodegeneration, and ischemic injuries. Since its emergence, many key components of the mammalian necroptotic pathway have been discovered including RIP1/3 kinases and MLKL, which together, form the core of the necrosome complex. RIP3-dependent phosphorylation of MLKL promotes the formation of high molecular weight MLKL polymers that disrupt the integrity of the plasma membrane leading to cell death. However, the mechanistic details of MLKL polymerization and its precise function in executing necroptosis remain poorly understood. To gain insight into this process, the crosslinking property of the specific MLKL inhibitor Necrosulfonamide (NSA) was exploited as a strategy for identifying novel MLKL interacting proteins. Immunoprecipitation of NSA crosslinked MLKL complexes followed by mass spectrometric analysis revealed Thioredoxin-1, an essential cytosolic thiol oxidoreductase, to be a potential MLKL regulator. Based on its ability to reduce disulfide bonds on specific protein targets, it was hypothesized that Thioredoxin-1 actively maintains MLKL in a reduced monomeric state to suppress the occurrence of spontaneous necroptosis. Therefore, genetic and pharmacological approaches were taken to perturb Thioredoxin-1 activity as a means for testing whether it would induce necroptosis in human cells. The data presented here demonstrates that inhibiting Thioreoxin-1 activity using the compound PX-12 promoted RIP3-dependent MLKL S358 phosphorylation, polymer formation, and caspase-independent necrotic cell death. Additionally, PX12-induced cell death was rescued by co-treating cells with NSA highlighting the involvement of MLKL in this cell death pathway. These results were corroborated by shRNA-mediated knockdown of Thioreoxin-1 mRNA. Altogether, these findings point to Thioredoxin-1 as a critical regulator of MLKL activity and necroptosis in human cells.