1,420 research outputs found

    The chaperone TRAP1 as a modulator of the mitochondrial adaptations in cancer cells

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    Mitochondria can receive, integrate, and transmit a variety of signals to shape many biochemical activities of the cell. In the process of tumor onset and growth, mitochondria contribute to the capability of cells of escaping death insults, handling changes in ROS levels, rewiring metabolism, and reprograming gene expression. Therefore, mitochondria can tune the bioenergetic and anabolic needs of neoplastic cells in a rapid and flexible way, and these adaptations are required for cell survival and proliferation in the fluctuating environment of a rapidly growing tumor mass. The molecular bases of pro-neoplastic mitochondrial adaptations are complex and only partially understood. Recently, the mitochondrial molecular chaperone TRAP1 (tumor necrosis factor receptor associated protein 1) was identified as a key regulator of mitochondrial bioenergetics in tumor cells, with a profound impact on neoplastic growth. In this review, we analyze these findings and discuss the possibility that targeting TRAP1 constitutes a new antitumor approach

    Strategies and Functional Consequences of Inhibiting Protein-Protein Interactions.

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    Networks of protein-protein interactions (PPIs) are essential in all aspects of cellular biology. At the nodes of these networks are multi-protein complexes that are often composed of dynamic, exchangeable modules assembled around a central enzyme. In this thesis, I have used the molecular chaperone heat shock protein 70 (Hsp70) as a model to develop ways of creating inhibitors of PPIs that tune the assembly and function of multi-protein complexes. Hsp70 is an ATPase and master regulator of protein homeostasis that interacts with co-chaperones, including nucleotide exchange factors (NEFs) and J-proteins. There is interest in creating chemical inhibitors that selectively interrupt PPIs between Hsp70 and its co-chaperones, as these molecules would be powerful chemical probes for validating Hsp70 as a target in cancer and other diseases. In this dissertation, I first review how advances in chemical screening methodologies, structural and computational biology, and proteomics have paved the way for the discovery of potent PPI inhibitors, even for difficult targets such as Hsp70 complexes. In Chapter 2, I develop a new high throughput screening (HTS) method in which Hsp70 is combined with co-chaperones and the ATPase activity of the combination is measured. I use this method to identify new inhibitors of Hsp70, characterizing their binding sites and molecular mechanism by NMR, mutagenesis and biochemical approaches. Importantly, I found that this HTS method reveals inhibitors of multiple PPIs within the Hsp70 system, including the interactions with NEFs and J proteins. This approach allowed me to find that Hsp70-NEF complexes control the stability of inhibitor of apoptosis (IAP) protein family members. In Chapter 3, I characterize IAPs as new “clients” of the Hsp70 system and explore the physical interaction between these proteins. That work establishes IAPs as the first sensitive, selective biomarkers suitable for use in pre-clinical studies of Hsp70 inhibitors. Finally, I show how inhibiting the Hsp70-NEF interaction has effects throughout the broader PPI network in Chapter 4. Together, these findings not only have important implications for Hsp70 drug discovery, but they also illustrate, more broadly, how small molecules can be used to re-shape multi-protein complexes and propagate changes throughout PPI networks.PHDChemical BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/135910/1/ceslaura_1.pd

    A dynamics based analysis of allosteric modulation in heat shock proteins

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    The 70 kDa and 90 kDa heat shock proteins (Hsp70 and Hsp90) are molecular chaperones that play central roles in maintaining cellular homeostasis in all organisms of life with the exception of archaea. In addition to their general chaperone function in protein quality control, Hsp70 and Hsp90 cooperate in the regulation and activity of some 200 known natively folded protein clients which include protein kinases, transcription factors and receptors, many of which are implicated as key regulators of essential signal transduction pathways. Both chaperones are considered to be large multi-domain proteins that rely on ATPase activity and co-chaperone interactions to regulate their conformational cycles for peptide binding and release. The unique positioning of Hsp90 at the crossroads of several fundamental cellular pathways coupled with its known association with diverse oncogenic peptide clients has brought the molecular chaperone under increasing interest as a potential anti-cancer target that is crucially implicated with all eight hallmarks of the disease. Current orthosteric drug discovery efforts aimed at the inhibition of the ATPase domain of Hsp90 have been limited due to high levels of associated toxicity. In an effort to circumnavigate this, the combined focus of research efforts is shifting toward alternative approaches such as interference with co-chaperone binding and the allosteric inhibition/activation of the molecular chaperone. The overriding aim of this thesis was to demonstrate how the computational technique of Perturbation response scanning (PRS) coupled with all-atom molecular dynamics simulations (MD) and dynamic residue interaction network (DRN) analysis can be used as a viable strategy to efficiently scan and accurately identify allosteric control element capable of modulating the functional dynamics of a protein. In pursuit of this goal, this thesis also contributes to the current understanding of the nucleotide dependent allosteric mechanisms at play in cellular functionality of both Hsp70 and Hsp90. All-atom MD simulations of E. coli DnaK provided evidence of nucleotide driven modulation of conformational dynamics in both the catalytically active and inactive states. PRS analysis employed on these trajectories demonstrated sensitivity toward bound nucleotide and peptide substrate, and provided evidence of a putative allosterically active intermediate state between the ATPase active and inactive conformational states. Simultaneous binding of ATP and peptide substrate was found to allosterically prime the chaperone for interstate conversion regardless of the transition direction. Detailed analysis of these allosterically primed states revealed select residue sites capable of selecting a coordinate shift towards the opposite conformational state. In an effort to validate these results, the predicted allosteric hot spot sites were cross-validated with known experimental works and found to overlap with functional sites implicated in allosteric signal propagation and ATPase activation in Hsp70. This study presented for the first time, the application of PRS as a suitable diagnostic tool for the elucidation and quantification of the allosteric potential of select residues to effect functionally relevant global conformational rearrangements. The PRS methodology described in this study was packaged within the Python programming environment in the MD-TASK software suite for command-line ease of use and made freely available. Homology modelling techniques were used to address the lack of experimental structural data for the human cytosolic isoform of Hsp90 and for the first time provided accurate full-length structural models of human Hsp90α in fully-closed and partially-open conformations. Long-range all-atom MD simulations of these structures revealed nucleotide driven modulation of conformational dynamics in Hsp90. Subsequent DRN and PRS analysis of these MD trajectories allowed for the quantification and elucidation of nucleotide driven allosteric modulation in the molecular chaperone. A detailed PRS analysis revealed allosteric inter-domain coupling between the extreme terminals of the chaperone in response to external force perturbations at either domain. Furthermore PRS also identified several individual residue sites that are capable of selecting conformational rearrangements towards functionally relevant states which may be considered to be putative allosteric target sites for future drug discovery efforts Molecular docking techniques were employed to investigate the modulation of conformational dynamics of human Hsp90α in response to ligand binding interactions at two identified allosteric sites at the C-terminal. High throughput screening of a small library of natural compounds indigenous to South Africa revealed three hit compounds at these sites: Cephalostatin 17, 20(29)-Lupene-3β isoferulate and 3'-Bromorubrolide F. All-atom MD simulations on these protein-ligand complexes coupled with DRN analysis and several advanced trajectory based analysis techniques provided evidence of selective allosteric modulation of Hsp90α conformational dynamics in response to the identity and location of the bound ligands. Ligands bound at the four-helix bundle presented as putative allosteric inhibitors of Hsp90α, driving conformational dynamics in favour of dimer opening and possibly dimer separation. Meanwhile, ligand interactions at an adjacent sub-pocket located near the interface between the middle and C-terminal domains demonstrated allosteric activation of the chaperone, modulating conformational dynamics in favour of the fully-closed catalytically active conformational state. Taken together, the data presented in this thesis contributes to the understanding of allosteric modulation of conformational dynamics in Hsp70 and Hsp90, and provides a suitable platform for future biochemical and drug discovery studies. Furthermore, the molecular docking and computational identification of allosteric compounds with suitable binding affinity for allosteric sites at the CTD of human Hsp90α provide for the first time “proof-of-principle” for the use of PRS in conjunction with MD simulations and DRN analysis as a suitable method for the rapid identification of allosteric sites in proteins that can be probed by small molecule interaction. The data presented in this section could pave the way for future allosteric drug discovery studies for the treatment of Hsp90 associated pathologies

    Mechanical Properties of Chaperone BiP, the Master Regulator of the Endoplasmic Reticulum

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    Immunoglobulin heavy-chain-binding protein (BiP protein) is a 75-kDa Hsp70 monomeric ATPase motor that plays broad and crucial roles maintaining proteostasis inside the cell. Its malfunction has been related with the appearance of many and important health problems such as neurodegenerative diseases, cancer, and heart diseases, among others. In particular, it is involved in many endoplasmic reticulum (ER) processes and functions, such as protein synthesis, folding, and assembly, and also it works in the posttranslational mechanism of protein translocation. However, it is unknown what kind of molecular motor BiP works like, since the mechanochemical mechanism that BiP utilizes to perform its work during posttranslational translocation across the ER is not fully understood. One novel approach to study both structural and catalytic properties of BiP considers that the viscoelastic regime behavior of the enzymes (considering them as a spring) and their mechanical properties are correlated with catalysis and ligand binding. Structurally, BiP is formed by two domains, and to establish a correlation between BiP structure and catalysis and how its conformational and viscoelastic changes are coupled to ligand binding, catalysis, and allosterism (information transmitted between the domains), optical tweezers and nano-rheology techniques have been essential in this regard

    Role of Hsp70 ATPase Domain Intrinsic Dynamics and Sequence Evolution in Enabling its Functional Interactions with NEFs

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    Catalysis of ADP-ATP exchange by nucleotide exchange factors (NEFs) is central to the activity of Hsp70 molecular chaperones. Yet, the mechanism of interaction of this family of chaperones with NEFs is not well understood in the context of the sequence evolution and structural dynamics of Hsp70 ATPase domains. We studied the interactions of Hsp70 ATPase domains with four different NEFs on the basis of the evolutionary trace and co-evolution of the ATPase domain sequence, combined with elastic network modeling of the collective dynamics of the complexes. Our study reveals a subtle balance between the intrinsic (to the ATPase domain) and specific (to interactions with NEFs) mechanisms shared by the four complexes. Two classes of key residues are distinguished in the Hsp70 ATPase domain: (i) highly conserved residues, involved in nucleotide binding, which mediate, via a global hinge-bending, the ATPase domain opening irrespective of NEF binding, and (ii) not-conserved but co-evolved and highly mobile residues, engaged in specific interactions with NEFs (e.g., N57, R258, R262, E283, D285). The observed interplay between these respective intrinsic (pre-existing, structure-encoded) and specific (co-evolved, sequence-dependent) interactions provides us with insights into the allosteric dynamics and functional evolution of the modular Hsp70 ATPase domain

    Chaperones and chaperone-substrate complexes: dynamic playgrounds for NMR spectroscopists

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    The majority of proteins depend on a well-defined three-dimensional structure to obtain their functionality. In the cellular environment, the process of protein folding is guided by molecular chaperones to avoid misfolding, aggregation, and the generation of toxic species. To this end, living cells contain complex networks of molecular chaperones, which interact with substrate polypeptides by a multitude of different functionalities: transport them towards a target location, help them fold, unfold misfolded species, resolve aggregates, or deliver them towards a proteolysis machinery. Despite the availability of high-resolution crystal structures of many important chaperones in their substrate-free apo forms, structural information about how substrates are bound by chaperones and how they are protected from misfolding and aggregation is very sparse. This lack of information arises from the highly dynamic nature of chaperone-substrate complexes, which so far has largely hindered their crystallization. This highly dynamic nature makes chaperone-substrate complexes good targets for NMR spectroscopy. Here, we review the results achieved by NMR spectroscopy to understand chaperone function in general and details of chaperone-substrate interactions in particular. We assess the information content and applicability of different NMR techniques for the characterization of chaperones and chaperone-substrate complexes. Finally, we highlight three recent studies, which have provided structural descriptions of chaperone-substrate complexes at atomic resolution

    The known unknowns of the Hsp90 chaperone

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    Molecular chaperones are vital proteins that maintain protein homeostasis by assisting in protein folding, activation, degradation, and stress protection. Among them, heat-shock protein 90 (Hsp90) stands out as an essential proteostasis hub in eukaryotes, chaperoning hundreds of "clients" (substrates). After decades of research, several "known unknowns" about the molecular function of Hsp90 remain unanswered, hampering rational drug design for the treatment of cancers, neurodegenerative and other diseases. We highlight three fundamental open questions, reviewing the current state of the field for each, and discuss new opportunities, including single-molecule technologies, to answer the known unknowns of the Hsp90 chaperone.Comment: 29 pages, 4 figure

    Regulation of Human Hsp70 by its Nucleotide Exchange Factors (NEFs).

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    Heat shock protein 70 (Hsp70) is an abundant and ubiquitous molecular chaperone that is responsible for maintenance of the human proteome. Accordingly, Hsp70 has become an attractive drug target for neurodegenerative and hyperproliferative disorders; however it is difficult to imagine strategies for inhibiting its pathobiology without impacting its essential roles. Fortunately, Hsp70 does not work alone, and instead employs a large network of co-chaperone proteins, which can tune Hsp70 activity and influence disease state. These co-chaperone proteins provide potential handles for targeting Hsp70 without disrupting overall proteostasis. One such class of co-chaperones proteins known as the Nucleotide Exchange Factors (NEFs), are a particular appealing target. NEFs bind Hsp70 and help to facilitate the exchange of ADP for ATP. The biochemistry of the NEF family of co-chaperones has classically been investigated using the prokaryotic NEF, GrpE, as a model. However, the eukaryotic cytosol does not contain a GrpE homolog. Rather, there are three main sub-classes of human NEFs: Hsp110, HspBP1, and the BAG proteins, all of which are structurally distinct with little sequence homology. Consistent with their diverse structures, they also differ in their mode of binding to Hsp70 and their roles in guiding Hsp70 biology. For example, BAG2 is associated with proteasomal degradation of the Hsp70 substrate, tau, while BAG1-Hsp70 is linked to increased tau stability. These observations suggest that the formation of specific NEF-Hsp70 complexes may help decide the fate of Hsp70-bound substrates. Additionally, these findings illustrate that differential disruption of specific Hsp70-NEF contacts might be beneficial in disease. In this thesis work I have systematically characterized the human Hsp70 NEFs, including how they interact with Hsp70, how the influence Hsp70 biochemistry and how they can bridge Hsp70 with other classes of chaperone proteins. I have used high throughput screening methods to search for chemical matter that can modulate Hsp70-NEF interactions, and we have shown that inhibitors of Hsp70-NEF interactions can be beneficial for treating disease. This thesis work has significantly advanced our knowledge of human Hsp70 regulation, and has provided groundwork for future studies on other Hsp70 co-chaperones and proteostasis components.PhDBiological ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/111611/1/rauchjn_1.pd

    Structure and Molecular Mechanism of ER Stress Signaling by the Unfolded Protein Response Signal Activator IRE1

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    The endoplasmic reticulum (ER) is an important site for protein folding and maturation in eukaryotes. The cellular requirement to synthesize proteins within the ER is matched by its folding capacity. However, the physiological demands or aberrations in folding may result in an imbalance which can lead to the accumulation of misfolded protein, also known as “ER stress.” The unfolded protein response (UPR) is a cell-signaling system that readjusts ER folding capacity to restore protein homeostasis. The key UPR signal activator, IRE1, responds to stress by propagating the UPR signal from the ER to the cytosol. Here, we discuss the structural and molecular basis of IRE1 stress signaling, with particular focus on novel mechanistic advances. We draw a comparison between the recently proposed allosteric model for UPR induction and the role of Hsp70 during polypeptide import to the mitochondrial matrix

    Modeling Signal Propagation Mechanisms and Ligand-Based Conformational Dynamics of the Hsp90 Molecular Chaperone Full-Length Dimer

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    Hsp90 is a molecular chaperone essential for protein folding and activation in normal homeostasis and stress response. ATP binding and hydrolysis facilitate Hsp90 conformational changes required for client activation. Hsp90 plays an important role in disease states, particularly in cancer, where chaperoning of the mutated and overexpressed oncoproteins is important for function. Recent studies have illuminated mechanisms related to the chaperone function. However, an atomic resolution view of Hsp90 conformational dynamics, determined by the presence of different binding partners, is critical to define communication pathways between remote residues in different domains intimately affecting the chaperone cycle. Here, we present a computational analysis of signal propagation and long-range communication pathways in Hsp90. We carried out molecular dynamics simulations of the full-length Hsp90 dimer, combined with essential dynamics, correlation analysis, and a signal propagation model. All-atom MD simulations with timescales of 70 ns have been performed for complexes with the natural substrates ATP and ADP and for the unliganded dimer. We elucidate the mechanisms of signal propagation and determine “hot spots” involved in interdomain communication pathways from the nucleotide-binding site to the C-terminal domain interface. A comprehensive computational analysis of the Hsp90 communication pathways and dynamics at atomic resolution has revealed the role of the nucleotide in effecting conformational changes, elucidating the mechanisms of signal propagation. Functionally important residues and secondary structure elements emerge as effective mediators of communication between the nucleotide-binding site and the C-terminal interface. Furthermore, we show that specific interdomain signal propagation pathways may be activated as a function of the ligand. Our results support a “conformational selection model” of the Hsp90 mechanism, whereby the protein may exist in a dynamic equilibrium between different conformational states available on the energy landscape and binding of a specific partner can bias the equilibrium toward functionally relevant complexes
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