Dana Reichmann

Order out of Disorder: Working Cycle of an Intrinsically Unfolded Chaperone

The redox-regulated chaperone Hsp33 protects organisms against oxidative stress that leads to protein unfolding. Activation of Hsp33 is triggered by the oxidative unfolding of its own redox-sensor domain, making Hsp33 a member of a recently discovered class of chaperones that require partial unfolding for full chaperone activity. Here we address the long-standing question of how chaperones recognize client proteins. We show that Hsp33 uses its own intrinsically disordered regions to discriminate between unfolded and partially structured folding intermediates. Binding to secondary structure elements in client proteins stabilizes Hsp33s intrinsically disordered regions, and this stabilization appears to mediate Hsp33s high affinity for structured folding intermediates. Return to nonstress conditions reduces Hsp33s disulfide bonds, which then significantly destabilizes the bound client proteins and in doing so converts them into less-structured, folding-competent client proteins of ATP-dependent foldases. We propose a model in which energy-independent chaperones use internal order-to-disorder transitions to control substrate binding and release.

Using Quantitative Redox Proteomics to Dissect the Yeast Redoxome

Background: Proteins with redox-sensitive thiols confer rapid response to changes in redox conditions and/or oxidant levels. Results: Quantitative redox proteomics unveiled steady-state thiol oxidation states of ∼5% of yeast proteins and revealed physiologically relevant redox- and peroxide-sensitive proteins in yeast. Conclusion: Redox-sensitive thiols appear structurally distinct from peroxide-sensitive thiols. Significance: Pathways are fine-tuned by protein oxidation under both non-stress and oxidative stress conditions. To understand and eventually predict the effects of changing redox conditions and oxidant levels on the physiology of an organism, it is essential to gain knowledge about its redoxome: the proteins whose activities are controlled by the oxidation status of their cysteine thiols. Here, we applied the quantitative redox proteomic method OxICAT to Saccharomyces cerevisiae and determined the in vivo thiol oxidation status of almost 300 different yeast proteins distributed among various cellular compartments. We found that a substantial number of cytosolic and mitochondrial proteins are partially oxidized during exponential growth. Our results suggest that prevailing redox conditions constantly control central cellular pathways by fine-tuning oxidation status and hence activity of these proteins. Treatment with sublethal H2O2 concentrations caused a subset of 41 proteins to undergo substantial thiol modifications, thereby affecting a variety of different cellular pathways, many of which are directly or indirectly involved in increasing oxidative stress resistance. Classification of the identified protein thiols according to their steady-state oxidation levels and sensitivity to peroxide treatment revealed that redox sensitivity of protein thiols does not predict peroxide sensitivity. Our studies provide experimental evidence that the ability of protein thiols to react to changing peroxide levels is likely governed by both thermodynamic and kinetic parameters, making predicting thiol modifications challenging and de novo identification of peroxide sensitive protein thiols indispensable.

Characterizing WW Domain Interactions of Tumor Suppressor WWOX Reveals Its Association with Multiprotein Networks

Background: WWOX encodes a 46-kDa tumor suppressor. Results: WW1 domain of WWOX mediates its protein-protein interaction with PY motifs that are involved in molecular processes, including transcription, RNA processing, and metabolism. Conclusion: The WW1 domain of WWOX provides a versatile platform that links WWOX with individual proteins associated with physiologically important networks. Significance: This study provides a better understanding of WWOX biology in normal and disease states. WW domains are small modules present in regulatory and signaling proteins that mediate specific protein-protein interactions. The WW domain-containing oxidoreductase (WWOX) encodes a 46-kDa tumor suppressor that contains two N-terminal WW domains and a central short-chain dehydrogenase/reductase domain. Based on its ligand recognition motifs, the WW domain family is classified into four groups. The largest one, to which WWOX belongs, recognizes ligands with a PPXY motif. To pursue the functional properties of the WW domains of WWOX, we employed mass spectrometry and phage display experiments to identify putative WWOX-interacting partners. Our analysis revealed that the first WW (WW1) domain of WWOX is the main functional interacting domain. Furthermore, our study uncovered well known and new PPXY-WW1-interacting partners and shed light on novel LPXY-WW1-interacting partners of WWOX. Many of these proteins are components of multiprotein complexes involved in molecular processes, including transcription, RNA processing, tight junction, and metabolism. By utilizing GST pull-down and immunoprecipitation assays, we validated that WWOX is a substrate of the E3 ubiquitin ligase ITCH, which contains two LPXY motifs. We found that ITCH mediates Lys-63-linked polyubiquitination of WWOX, leading to its nuclear localization and increased cell death. Our data suggest that the WW1 domain of WWOX provides a versatile platform that links WWOX with individual proteins associated with physiologically important networks.

Computational redesign of a protein-protein interface for high affinity and binding specificity using modular architecture and naturally occurring template fragments.

A new method is presented for the redesign of protein-protein interfaces, resulting in specificity of the designed pair while maintaining high affinity. The design is based on modular interface architecture and was carried out on the interaction between TEM1 beta-lactamase and its inhibitor protein, beta-lactamase inhibitor protein. The interface between these two proteins is composed of several mostly independent modules. We previously showed that it is possible to delete a complete module without affecting the overall structure of the interface. Here, we replace a complete module with structure fragments taken from nonrelated proteins. Nature-optimized fragments were chosen from 10(7) starting templates found in the Protein Data Bank. A procedure was then developed to identify sets of interacting template residues with a backbone arrangement mimicking the original module. This generated a final list of 361 putative replacement modules that were ranked using a novel scoring function based on grouped atom-atom contact surface areas. The top-ranked designed complex exhibited an affinity of at least the wild-type level and a mode of binding that was remarkably specific despite the absence of negative design in the procedure. In retrospect, the combined application of three factors led to the success of the design approach: utilizing the modular construction of the interface, capitalizing on native rather than artificial templates, and ranking with an accurate atom-atom contact surface scoring function.

Time line of redox events in aging postmitotic cells

The precise roles that oxidants play in lifespan and aging are still unknown. Here, we report the discovery that chronologically aging yeast cells undergo a sudden redox collapse, which affects over 80% of identified thiol-containing proteins. We present evidence that this redox collapse is not triggered by an increase in endogenous oxidants as would have been postulated by the free radical theory of aging. Instead it appears to be instigated by a substantial drop in cellular NADPH, which normally provides the electron source for maintaining cellular redox homeostasis. This decrease in NADPH levels occurs very early during lifespan and sets into motion a cascade that is predicted to down-regulate most cellular processes. Caloric restriction, a near-universal lifespan extending measure, increases NADPH levels and delays each facet of the cascade. Our studies reveal a time line of events leading up to the system-wide oxidation of the proteome days before cell death. DOI: http://dx.doi.org/10.7554/eLife.00306.001

A Quantitative, Real-Time Assessment of Binding of Peptides and Proteins to Gold Surfaces

Interactions of peptides and proteins with inorganic surfaces are important to both natural and artificial systems; however, a detailed understanding of such interactions is lacking. In this study, we applied new approaches to quantitatively measure the binding of amino acids and proteins to gold surfaces. Real-time surface plasmon resonance (SPR) measurements showed that TEM1-β-lactamase inhibitor protein (BLIP) interacts only weakly with Au nanoparticles (NPs). However, fusion of three histidine residues to BLIP (3H-BLIP) resulted in a significant increase in the binding to the Au NPs, which further increased when the histidine tail was extended to six histidines (6H-BLIP). Further increasing the number of His residues had no effect on the binding. A parallel study using continuous (111)-textured Au surfaces and single-crystalline, (111)-oriented, Au islands by ellipsometry, FTIR, and localized surface plasmon resonance (LSPR) spectroscopy further confirmed the results, validating the broad applicability of Au NPs as model surfaces. Evaluating the binding of all other natural amino acid homotripeptides fused to BLIP (except Cys and Pro) showed that aromatic and positively-charged residues bind preferentially to Au with respect to small aliphatic and negatively charged residues, and that the rate of association is related to the potency of binding. The binding of all fusions was irreversible. These findings were substantiated by SPR measurements of synthesized, free, soluble tripeptides using Au-NP-modified SPR chips. Here, however, the binding was reversible allowing for determination of binding affinities that correlate with the binding potencies of the related BLIP fusions. Competition assays performed between 3H-BLIP and the histidine tripeptide (3 His) suggest that Au binding residues promote the adsorption of proteins on the surface, and by this facilitate the irreversible interaction of the polypeptide chain with Au. The binding of amino acids to Au was simulated by using a continuum solvent model, showing agreement with the experimental values. These results, together with the observed binding potencies and kinetics of the BLIP fusions and free peptides, suggest a binding mechanism that is markedly different from biological protein-protein interactions.

Oxidative stress and Redox regulation

The thiol functional group of the amino acid cysteine can undergo a wide array of oxidative modifications and perform a countless number of physiological functions. In addition to forming covalent cross-links that stabilize protein structure and functioning as a powerful nucleophile in many enzyme active sites, cysteine appears to be the principal actor in redox signaling, functioning as a regulatory reversible molecular switch. It is increasingly appreciated that the thiol group of cysteine in subset of proteins undergoes oxidative modification in response to changes in the intracellular redox environment. To understand these complex but critical biological phenomena, the chemistry of the thiol functionality and related oxidation products must also be taken into consideration. Selective methods to monitor and quantify discrete cysteine modifications will be central to understanding their regulatory and pathophysiologic function. Accordingly, this chapter focuses on the chemical feature of thiol oxidation and on selective methods for detecting oxidants and individual cysteine chemotypes.

Unfolding of Metastable Linker Region Is at the Core of Hsp33 Activation as a Redox-regulated Chaperone

Hsp33, a molecular chaperone specifically activated by oxidative stress conditions that lead to protein unfolding, protects cells against oxidative protein aggregation. Stress sensing in Hsp33 occurs via its C-terminal redox switch domain, which consists of a zinc center that responds to the presence of oxidants and an adjacent metastable linker region, which responds to unfolding conditions. Here we show that single mutations in the N terminus of Hsp33 are sufficient to either partially (Hsp33-M172S) or completely (Hsp33-Y12E) abolish this post-translational regulation of Hsp33 chaperone function. Both mutations appear to work predominantly via the destabilization of the Hsp33 linker region without affecting zinc coordination, redox sensitivity, or substrate binding of Hsp33. We found that the M172S substitution causes moderate destabilization of the Hsp33 linker region, which seems sufficient to convert the redox-regulated Hsp33 into a temperature-controlled chaperone. The Y12E mutation leads to the constitutive unfolding of the Hsp33 linker region thereby turning Hsp33 into a constitutively active chaperone. These results demonstrate that the redox-controlled unfolding of the Hsp33 linker region plays the central role in the activation process of Hsp33. The zinc center of Hsp33 appears to act as the redox-sensitive toggle that adjusts the thermostability of the linker region to the cell redox status. In vivo studies confirmed that even mild overexpression of the Hsp33-Y12E mutant protein inhibits bacterial growth, providing important evidence that the tight functional regulation of Hsp33 chaperone activity plays a vital role in bacterial survival.

Similar chemistry, but different bond preferences in inter versus intra-protein interactions

Proteins fold into a well‐defined structure as a result of the collapse of the polypeptide chain, while transient protein‐complex formation mainly is a result of binding of two folded individual monomers. Therefore, a protein–protein interface does not resemble the core of monomeric proteins, but has a more polar nature. Here, we address the question of whether the physico‐chemical characteristics of intraprotein versus interprotein bonds differ, or whether interfaces are different from folded monomers only in the preference for certain types of interactions. To address this question we assembled a high resolution, nonredundant, protein–protein interaction database consisting of 1374 homodimer and 572 heterodimer complexes, and compared the physico‐chemical properties of these interactions between protein interfaces and monomers. We performed extensive statistical analysis of geometrical properties of interatomic interactions of different types: hydrogen bonds, electrostatic interactions, and aromatic interactions. Our study clearly shows that there is no significant difference in the chemistry, geometry, or packing density of individual interactions between interfaces and monomeric structures. However, the distribution of different bonds differs. For example, side‐chain–side‐chain interactions constitute over 62% of all interprotein interactions, while they make up only 36% of the bonds stabilizing a protein structure. As on average, properties of backbone interactions are different from those of side chains, a quantitative difference is observed. Our findings clearly show that the same knowledge‐based potential can be used for protein‐binding sites as for protein structures. However, one has to keep in mind the different architecture of the interfaces and their unique bond preference. Proteins 2008.

The roles of conditional disorder in redox proteins

Cells are constantly exposed to various oxidants, either generated endogenously due to metabolic activity or exogenously. One way that cells respond to oxidants is through the action of redox-regulated proteins. These proteins also play important roles in oxidant signaling and protein biogenesis events. The key sensors built into redox-regulated proteins are cysteines, which undergo reversible thiol oxidation in response to changes in the oxidation status of the cellular environment. In this review, we discuss three examples of redox-regulated proteins found in bacteria, mitochondria, and chloroplasts. These proteins use oxidation of their redox-sensitive cysteines to reversibly convert large structural domains into more disordered regions or vice versa. These massive structural rearrangements are directly implicated in the functions of these proteins.