Computational Studies on the Relation Between Macromolecular Dynamics and Protein Binding and Function

Computational methods can help to better understand and analyze the interaction of proteins and their binding partners. This interaction is influenced by many factors, including specific sequence variants, the dynamics and electrostatics of the proteins, as well as further physicochemical properties of the corresponding binding partners. A detailed investigation of these different, and often complicated, properties helps to better understand the functionality of proteins, for which the interaction with other molecules plays a crucial role. The work presented here provides new methodologies, implemented in webservers and software, which assist during the analysis of proteins. Furthermore, in an application case, computational methods and analyses in combination with experimental results were used to detect a specific interaction network of proteins. The new ProSAT+ webserver enables the visualization of protein sequence annotations in the context of the three–dimensional protein structure and contains additional options for visualizing and sharing protein annotations. The sequence information allows an easy, but extensive analysis of proteins. The functionality of the ProSAT+ webserver can be integrated into other webservers, which was done in the case of the two other webservers for the analysis of protein binding pockets described here. A tool for the LigDig webserver was developed that provides the comparison of protein binding pockets by the alignment and visualization of the binding pockets based on an existing algorithm. The new TRAPP webserver assists in the analysis of protein binding pocket dynamics. The existing TRAPP software was used, and a user web interface was implemented to simplify the usability. Additional new functionalities were also developed, such as the visualization of protein sequence conservation in context of all other TRAPP results in the three–dimensional structure. This allows the detection of conserved or non–conserved regions inside the binding pocket, which might influence the dynamics of the pocket. This newly gained information can be used during the process of designing selective inhibitors. During the protein disaggregation process, members from different classes of the so-called J–protein (HSP40) co–chaperones play a crucial role. The synergetic application of different computational methods and experiments enabled the detection of an interclass specific J–protein interaction and indicated that the interaction evolved to enable a high efficiency in the disaggregation process. The resulting data of performed protein domain docking simulations required an update of the standard clustering workflow. This new methodology can be applied for protein docking in cases that have problems with multiple, weakly specific interaction sites. The work presented here facilitates in many ways the analysis of proteins, including their structure and sequence features, as well as, their dynamics and interactions with their binding partners. The new methods are provided as webservers and therefore are accessible, and easy to use for all researchers. This can assist in many research projects and provide relevant information. The analyses of the J–proteins improved the knowledge about their biological role and functionality, and therefore provide an important contribution for a better understanding of the overall protein disaggregation process

Impact of carbonylation on glutathione peroxidase-1 activity in human hyperglycemic endothelial cells

Aims: High levels of glucose and reactive carbonyl intermediates of its degradation pathway such as methylglyoxal (MG) may contribute to diabetic complications partly via increased generation of reactive oxygen species (ROS). This study focused on glutathione peroxidase-1 (GPx1) expression and the impact of carbonylation as an oxidative protein modification on GPx1 abundance and activity in human umbilical vein endothelial cells (HUVEC) under conditions of mild to moderate oxidative stress. Results: High extracellular glucose and MG enhanced intracellular ROS formation in HUVECs. Protein carbonylation was only transiently augmented pointing to an effective antioxidant defense in these cells. Nitric oxide synthase expression was decreased under hyperglycemic conditions but increased upon exposure to MG, whereas superoxide dismutase expression was not significantly affected. Increased glutathione peroxidase (GPx) activity seemed to compensate for a decrease in GPx1 protein due to enhanced degradation via the proteasome. Mass spectrometry analysis identified Lys-114 as a possible carbonylation target which provides a vestibule for the substrate H2O2 and thus enhances the enzymatic reaction. Innovation: Oxidative protein carbonylation has so far been associated with functional inactivation of modified target proteins mainly contributing to aging and age-related diseases. Here, we demonstrate that mild oxidative stress and subsequent carbonylation seem to activate protective cellular redox signaling pathways whereas severe oxidative stress overwhelms the cellular antioxidant defense leading to cell damage. Conclusions: This study may contribute to a better understanding of redox homeostasis and its role in the development of diabetes and related vascular complications. Keywords: Protein carbonylation, Reactive oxygen species, Hyperglycemia, Glutathione peroxidase-1, Endothelial cell

Evolution of an intricate J-protein network driving protein disaggregation in eukaryotes

Hsp70 participates in a broad spectrum of protein folding processes extending from nascent chain folding to protein disaggregation. This versatility in function is achieved through a diverse family of J-protein cochaperones that select substrates for Hsp70. Substrate selection is further tuned by transient complexation between different classes of J-proteins, which expands the range of protein aggregates targeted by metazoan Hsp70 for disaggregation. We assessed the prevalence and evolutionary conservation of J-protein complexation and cooperation in disaggregation. We find the emergence of a eukaryote-specific signature for interclass complexation of canonical J-proteins. Consistently, complexes exist in yeast and human cells, but not in bacteria, and correlate with cooperative action in disaggregation in vitro. Signature alterations exclude some J-proteins from networking, which ensures correct J-protein pairing, functional network integrity and J-protein specialization. This fundamental change in J-protein biology during the prokaryote-to-eukaryote transition allows for increased fine-tuning and broadening of Hsp70 function in eukaryotes

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opportunity for the controlled study o

Crucial HSP70 co-chaperone complex unlocks metazoan protein disaggregation

Protein aggregates are the hallmark of stressed and ageing cells, and characterize several pathophysiological states1, 2. Healthy metazoan cells effectively eliminate intracellular protein aggregates3, 4, indicating that efficient disaggregation and/or degradation mechanisms exist. However, metazoans lack the key heat-shock protein disaggregase HSP100 of non-metazoan HSP70-dependent protein disaggregation systems5, 6, and the human HSP70 system alone, even with the crucial HSP110 nucleotide exchange factor, has poor disaggregation activity in vitro4, 7. This unresolved conundrum is central to protein quality control biology. Here we show that synergic cooperation between complexed J-protein co-chaperones of classes A and B unleashes highly efficient protein disaggregation activity in human and nematode HSP70 systems. Metazoan mixed-class J-protein complexes are transient, involve complementary charged regions conserved in the J-domains and carboxy-terminal domains of each J-protein class, and are flexible with respect to subunit composition. Complex formation allows J-proteins to initiate transient higher order chaperone structures involving HSP70 and interacting nucleotide exchange factors. A network of cooperative class A and B J-protein interactions therefore provides the metazoan HSP70 machinery with powerful, flexible, and finely regulatable disaggregase activity and a further level of regulation crucial for cellular protein quality control