Protein aggregation is involved in a variety of diseases, including neurodegenerative diseases and cancer. The cellular environment is crowded by a plethora of cosolutes comprising small molecules and biomacromolecules at high concentrations, which may influence the aggregation of proteins in vivo. To account for the effect of cosolutes on cancer-related protein aggregation, we studied their effect on the aggregation of the cancer-related L106R mutant of the Axin protein. Axin is a key player in the Wnt signaling pathway, and the L106R mutation in its RGS domain results in a native molten globule that tends to form native-like aggregates. This results in uncontrolled activation of the Wnt signaling pathway, leading to cancer. We monitored the aggregation process of Axin RGS L106Rin vitro in the presence of a wide ensemble of cosolutes including polyols, amino acids, betaine and polyethylene glycol (PEG) crowders. Except myo-inositol, all polyols decreased RGS L106R aggregation, with carbohydrates exerting the strongest inhibition. Conversely, betaine and PEGs enhanced aggregation. These results are consistent with the reported effects of osmolytes and crowders on the stability of molten globular proteins and with both amorphous and amyloid aggregation mechanisms. We suggest a model of Axin L106R aggregation in vivo, whereby molecularly small osmolytes keep the protein as a free solublemolecule but the increased crowding of the bound state by macromolecules induces its aggregation at the nano-scale. Our study sheds light on the potential contribution of cosolutes to the onset of cancer as a protein misfolding disease, and on the relevance of aggregation in the molecular aetiology of cancer.​

Na⁺/H⁺ antiporters comprise a super-family (CPA) of membrane proteins that are found in all kingdoms of life and are essential in cellular homeostasis of pH, Na⁺ and volume. Their activity is strictly dependent on pH, a property that underpins their role in pH homeostasis. While several human homologues have long been drug targets, NhaA of Escherichia coli has become the paradigm for this class of secondary active transporters as NhaA crystal structure provided insight into the architecture of this molecular machine. However, the mechanism of the strict pH dependence of NhaA is missing. Here, as a follow up of a recent evolutionary analysis that identified a ‘CPA motif’, we rationally designed three E. coli NhaA mutants: D133S, I134T, and the double mutant D133S-I134T. Exploring growth phenotype, transport activity and Li⁺-binding of the mutants, we revealed that Asp133 does not participate directly in proton binding, nor does it directly dictate the pH-dependent transport of NhaA. Strikingly, the variant I134T lost some of the pH control, and the D133S-Il134T double mutant retained Li⁺ binding in a pH independent fashion. Concurrent to loss of pH control, these mutants bound Li⁺ more strongly than the WT. Both positions are in close vicinity to the ion-binding site of the antiporter, attributing the results to electrostatic interaction between these residues and Asp164 of the ion-binding site. This is consistent with pH sensing resulting from direct coupling between cation binding and deprotonation in Asp164, which applies also to other CPA antiporters that are involved in human diseases.

Unraveling the role of post-translational modification (PTM) patterns is one of the most urgent and unresolved issues facing the scientific community. Attempts to crack the phosphorylation bio-barcode led to significant findings, which suggest that many proteins cannot be regarded as a single entity but exist as several forms which differ in their phosphorylation patterns and their functions. While protein regions that do not contain PTMs can be rather simply mimicked using peptide libraries, heavily phosphorylated regions are much harder to study using the same tools. The differences between the syntheses of simple mono-, di- and tri-phosphopeptides and the synthesis of multiphosphopeptides are dramatic. While simple phosphopeptides can be synthesized using almost standard SPPS strategies, the synthesis of multiphosphopeptides is to date a major synthetic challenge. Synthesis of multiphosphopeptides requires the insertion of several phosphate groups simultaneously or sequentially into various positions on the peptide in the presence of many other potential modification sites. These groups are bulky, unstable and cannot be easily introduced when in close proximity. Moreover, since the same protein region can possess many alternative multiphosphorylation patterns, libraries comprising a large number of peptides with different degrees and positions of phosphorylation are essential. Many strategies have been developed to provide routes to enable the preparation of multiphosphopeptides. These methods are essentially different from the methods used for the preparation of simple phosphopeptides. In this review, we specifically emphasize the challenges and importance of synthesizing multiphosphopeptides and their libraries. The historical perspective and state of the art strategies are described. We demonstrate here how the different synthetic approaches attempt to address the special problems associated with the synthesis of multiphosphopeptides. The advantages and disadvantages of each strategy are discussed in order to provide a roadmap for the synthesis of such libraries. An overview of the existing strategies and some comments regarding future directions are provided. Applications of multiphosphopeptide libraries as tools to study the effect of phosphorylation patterns on the biological function of proteins are also described.

We describe a molecular characterization of the interaction between the cancer‐related proteins WWOX and p73. This interaction is mediated by the first of two WW domains (WW1) of WWOX and a PPXY‐motif‐containing region in p73. While phosphorylation of Tyr33 of WWOX and association with p73 are known to affect apoptotic activity, the quantitative effect of phosphorylation on this specific interaction is determined here for the first time. Using ITC and fluorescence anisotropy, we measured the binding affinity between WWOX domains and a p73 derived peptide, and showed that this interaction is regulated by Tyr phosphorylation of WW1. Chemical synthesis of the phosphorylated domains of WWOX revealed that the binding affinity of WWOX to p73 is decreased when WWOX is phosphorylated. This result suggests a fine‐tuning of binding affinity in a differential, ligand‐specific manner: the decrease in binding affinity of WWOX to p73 can free both partners to form new interactions.

Cyclic peptide-peptoid hybrids possess improved stability and selectivity over linear peptides and are thus better drug candidates. However, their synthesis is far from trivial and is usually difficult to automate. Here we describe a new rapid and efficient approach for the synthesis of click-based cyclic peptide-peptoid hybrids. Our methodology is based on a combination between easily synthesized building blocks, automated microwave assisted solid phase synthesis and bioorthogonal click cyclization. We proved the concept of this method using the INS peptide, which we have previously shown to activate the HIV-1 integrase enzyme. This strategy enabled the rapid synthesis and biophysical evaluation of a library of cyclic peptide-peptoid hybrids derived from HIV-1 integrase in high yield and purity. The new cyclic hybrids showed improved biological activity and were significantly more stable than the original linear INS peptide.

Intrinsically disordered regions in proteins (IDRs) mediate many disease‐related protein–protein interactions. However, the unfolded character and continuous conformational changes of IDRs make them difficult to target for therapeutic purposes. Here, we show that a designed peptide based on the disordered p53 linker domain can be used to target a partner IDR from the anti‐apoptotic iASPP protein, promoting apoptosis of cancer cells. The p53 linker forms a hairpin‐like structure with its two termini in close proximity. We designed a peptide derived from the disordered termini without the hairpin, designated as p53 LinkTer. The LinkTer peptide binds the disordered RT loop of iASPP with the same affinity as the parent p53 linker peptide, and inhibits the p53–iASPP interaction in vitro. The LinkTer peptide shows increased stability to proteolysis, penetrates cancer cells, causes nuclei shrinkage, and compromises the viability of cells. We conclude that a designed peptide comprising only the IDR from a peptide sequence can serve as an improved inhibitor since it binds its target protein without the need for pre‐folding, paving the way for therapeutic targeting of IDRs. See our cover profile.

Suppression of apoptosis is a key Hallmark of cancer cells, and reactivation of apoptosis is a major avenue for cancer therapy. We reveal an interaction between the two anti-apoptotic proteins iASPP and NAF-1, which are overexpressed in many types of cancer cells and tumors. iASPP is an inhibitory member of the ASPP protein family, whereas NAF-1 belongs to the NEET 2Fe–2S protein family. We show that the two proteins are stimulated to interact in cells during apoptosis. Using peptide array screening and computational methods we mapped the interaction interfaces of both proteins to residues 764–778 of iASPP that bind to a surface groove of NAF-1. A peptide corresponding to the iASPP 764–780 sequence stabilized the NAF-1 cluster, inhibited NAF-1 interaction with iASPP, and inhibited staurosporine-induced apoptosis activation in human breast cancer, as well as in PC-3 prostate cancer cells in which p53 is inactive. The iASPP 764–780 IC₅₀ value for inhibition of cell death in breast cancer cells was 13 ± 1 μM. The level of cell death inhibition by iASPP 764–780 was altered in breast cancer cells expressing different levels and/or variants of NAF-1, indicating that the peptide activity is associated with NAF-1 function. We propose that the interaction between iASPP and NAF-1 is required for apoptosis activation in cancer cells. This interaction uncovers a new layer in the highly complex regulation of cell death in cancer cells and opens new avenues of exploration into the development of novel anticancer drugs that reactivate apoptosis in malignant tumors.

Cardiolipin (CL) was shown to bound to the dimer interface of NhaA Na⁺/H⁺ antiporter. Here, we explore the cardiolipin-NhaA interaction both in vitro and in vivo. Using a novel and straightforward in-vitro assay in which n-dodecyl β-D maltoside (DDM) detergent is used to delipidate the dimer interface and to split the dimers into monomers; the monomers are subsequently exposed to cardiolipin or the other E. coli phospholipids. Most efficient reconstitution of dimers is observed by cardiolipin. This assay is likely to be applicable to future studies of protein–lipid interactions. In-vivo experiments further reveal that cardiolipin is necessary for NhaA survival. Although less efficient phosphatidyl-glycerol (PG) can also reconstitute NhaA monomers to dimers. We also identify a putative cardiolipin binding site. Our observations may contribute to drug design, as human NhaA homologues, which are involved in severe pathologies, might also require specific phospholipids.

Cellular functions of arrestins are determined in part by the pattern of phosphorylation on the G protein-coupled receptors (GPCRs) to which arrestins bind. Despite high-resolution structural data of arrestins bound to phosphorylated receptor C-termini, the functional role of each phosphorylation site remains obscure. Here, we employ a library of synthetic phosphopeptide analogues of the GPCR rhodopsin C-terminus and determine the ability of these peptides to bind and activate arrestins using a variety of biochemical and biophysical methods. We further characterize how these peptides modulate the conformation of arrestin-1 by nuclear magnetic resonance (NMR). Our results indicate different functional classes of phosphorylation sites: ‘key sites’ required for arrestin binding and activation, an ‘inhibitory site’ that abrogates arrestin binding, and ‘modulator sites’ that influence the global conformation of arrestin. These functional motifs allow a better understanding of how different GPCR phosphorylation patterns might control how arrestin functions in the cell.

Protein phosphorylation barcodes, clusters of several phosphorylation sites within a short unfolded region, control many cellular processes. Existing biochemical methods used to study the roles of these barcodes suffer from low selectivity and provide only qualitative data. Chemically synthesized multiphosphopeptides libraries are selective and specific, but their synthesis is extremely difficult using the current peptide synthesis methods. Here we decribe a new microwave assisted approach for synthesizing a library of multiphosphopeptides, using the C-terminus of rhodopsin as a proof of concept. Our approch utilizes multiple protocols for synthesizing libraries of multiphosphopeptides instead of the inefficent single protocol methods curretnly used. Using our approach we demonstrated the synthesis of with up to seven phosphorylated amino acids, sometimes next to each other, an accomplishment that was impractical before. Synthesizing the Rhodopsin derived multiphosphopeptide library enabled dissecting the precise phosphorylation barcode required for the recruitment, activation and modulation of the conformation of Arrestin. Since phosphorylation barcodes modulate the activity of hundreds of GPCRs, synthesizing libraries of multiphosphopeptides is the method of choice for studying their molecular mechanisms of action. Our approach provides an invaluable tool for evaluating how protein phosphorylation barcodes regulate their activity.

Multi-angle light scattering coupled with size exclusion chromatography (SEC-MALS) is a standard and common approach for characterizing protein mass, overall shape, aggregation, oligomerization, interactions and purity. The limited resolution of analytical SEC restricts in some instances the accurate analysis that can be accomplished by MALS. These include mixtures of protein populations with identical or very similar molecular masses, oligomers with poor separation and short peptides. Here we show that combining MALS with the higher resolution separation technique ion exchange (IEX-MALS) can allow precise analyses of samples that cannot be resolved by SEC-MALS. We conclude that IEX-MALS is a valuable and complementary method for protein characterization, especially for protein systems that could not be fully analyzed by SEC-MALS.

Na⁺/H⁺ antiporters have a crucial role in pH and Na⁺ homeostasis in cells. The crystal structure of NhaA, the main antiporter of Escherichia coli, has provided general insights into antiporter mechanisms and revealed a previously unknown structural fold, which has since been identified in several secondary active transporters. This unique structural fold is very delicately electrostatically balanced. Asp133 and Lys 300 have been ascribed essential roles in this balance and, more generally, in the structure and function of the antiporter. In this work, we show the multiple roles of Asp133 in NhaA: (i) The residue's negative charge is critical for the stability of the NhaA structure. (ii) Its main chain is part of the active site. (iii) Its side chain functions as an alkaline-pH-dependent gate, changing the protein's conformation from an inward-facing conformation at acidic pH to an outward-open conformation at alkaline pH, opening the periplasm funnel. On the basis of the experimental data, we propose a tentative mechanism integrating the structural and functional roles of Asp133.

Intrinsically disordered regions (IDRs) in proteins are highly abundant, but they are still commonly viewed as long stretches of polar, solvent accessible residues. Here we show that the disordered C-terminal domain of HIV-1 Rev has two sub-regions that carry out two distinct complementary roles of regulating oligomerization and contributing to stability. We propose this is carried out by a delicate balance between charged and hydrophobic residues within the IDR. We suggest that intrinsically disordered regions in proteins should be divided to sub domains similarly to structured regions, rather than being viewed as a long flexible stretches. This implicates that mutations in IDRs can affect protein function in disease just like known mutations in structured regions.

The p53 tumor suppressor protein is the most well studied as a regulator of transcription in the nucleus, where it exists primarily as a tetramer. However, there are other oligomeric states of p53 that are relevant to its regulation and activities. In unstressed cells, p53 is normally held in check by MDM2 that targets p53 for transcriptional repression, proteasomal degradation, and cytoplasmic localization. Here we discovered a hydrophobic region within the MDM2 N-terminal domain that binds exclusively to the dimeric form of the p53 C-terminal domain in vitro. In cell-based assays, MDM2 exhibits superior binding to, hyperdegradation of, and increased nuclear exclusion of dimeric p53 when compared with tetrameric wild-type p53. Correspondingly, impairing the hydrophobicity of the newly identified N-terminal MDM2 region leads to p53 stabilization. Interestingly, we found that dimeric mutant p53 is partially unfolded and is a target for ubiquitin-independent degradation by the 20S proteasome. Finally, forcing certain tumor-derived mutant forms of p53 into dimer configuration results in hyperdegradation of mutant p53 and inhibition of p53-mediated cancer cell migration. Gaining insight into different oligomeric forms of p53 may provide novel approaches to cancer therapy.

We have recently reported the covalent inhibition of HIV-1 integrase by an N-terminal succinimide-modified lens epithelium-derived growth factor (361-370) peptide. We also showed that this peptide is proteolytically stable. Here, we show that this inhibitor is stored as fibrils that serve as a stock for the inhibitory monomers. The fibrils increase the local concentration of the peptide at the target protein. When the monomers bind integrase, the equilibrium between the fibrils and their monomers shifts towards the formation of peptide monomers. The combination of fibril formation and subsequent proteolytic stability of the peptide may bring to new strategy for developing therapeutic agents.

G protein-coupled receptors (GPCRs) constitute the largest family of cell surface receptors. They can exist and act as dimers, but the requirement of dimers for agonist-induced signal initiation and structural dynamics remains largely unknown. Frizzled 6 (FZD₆) is a member of Class F GPCRs, which bind WNT proteins to initiate signaling. Here, we show that FZD₆dimerizes and that the dimer interface of FZD₆ is formed by the transmembrane α-helices four and five. Most importantly, we present the agonist-induced dissociation/re-association of a GPCR dimer through the use of live cell imaging techniques. Further analysis of a dimerization-impaired FZD₆ mutant indicates that dimer dissociation is an integral part of FZD₆ signaling to extracellular signal-regulated kinases1/2. The discovery of agonist-dependent dynamics of dimers as an intrinsic process of receptor activation extends our understanding of Class F and other dimerizing GPCRs, offering novel targets for dimer-interfering small molecules.Frizzled 6 (FZD₆) is a G protein-coupled receptor (GPCR) involved in several cellular processes. Here, the authors use live cell imaging and spectroscopy to show that FZD₆ forms dimers, whose association is regulated by WNT proteins and that dimer dissociation is crucial for FZD₆ signaling.

SCOPE:

Brown adipose tissue (BAT) is the main regulator of thermogenesis by increasing energy expenditure through the uncoupling of oxidative metabolism from ATP synthesis. There is a growing body of evidence for BAT being the key responsible organ in combating obesity and its related disorders. Herein we propose the fungal protein ostreolysin (Oly), which has been previously shown to bind to cholesterol-enriched raft-like membrane domains (lipid rafts) of mammalian cells, as a suitable candidate for interaction with brown preadipocytes. The aim of the present study was therefore to characterize the mechanism by which a recombinant version of ostreolysin (rOly) induces brown adipocyte differentiation.

METHODS AND RESULTS:

Primary isolated brown preadipocytes or HIB-1B brown preadipocyte cells were treated with rOly and the effects on morphology, lipid accumulation, respiration rate, and associated gene and protein expression were measured. rOly upregulated mRNA and protein levels of factors related to brown adipocyte differentiation, induced lipid droplet formation, and increased cellular respiration rate due to expression of uncoupling protein 1. rOly also upregulated β-tubulin expression, and therefore microtubules might be involved in its mechanism of action.

CONCLUSION:

rOly promotes brown adipocyte differentiation, suggesting a new mechanism for rOly's contribution to the battle against obesity.

AIMS:

A recently discovered group of conditionally disordered chaperones share a very unique feature; they need to lose structure to become active as chaperones. This activation mechanism makes these chaperones particularly suited to respond to protein-unfolding stress conditions, such as oxidative unfolding. However, the role of this disorder in stress-related activation, chaperone function, and the crosstalk with other chaperone systems is not yet clear. Here, we focus on one of the members of the conditionally disordered chaperones, a thiol-redox switch of the bacterial proteostasis system, Hsp33.

RESULTS:

By modifying the Hsp33's sequence, we reveal that the metastable region has evolved to abolish redox-dependent chaperone activity, rather than enhance binding affinity for client proteins. The intrinsically disordered region of Hsp33 serves as an anchor for the reduced, inactive state of Hsp33, and it dramatically affects the crosstalk with the synergetic chaperone system, DnaK/J. Using mass spectrometry, we describe the role that the metastable region plays in determining client specificity during normal and oxidative stress conditions in the cell. Innovation and Conclusion: We uncover a new role of protein plasticity in Hsp33's inactivation, client specificity, crosstalk with the synergistic chaperone system DnaK/J, and oxidative stress-specific interactions in bacteria. Our results also suggest that Hsp33 might serve as a member of the house-keeping proteostasis machinery, tasked with maintaining a "healthy" proteome during normal conditions, and that this function does not depend on the metastable linker region.

Binding of metal ions is an important regulatory mechanism in proteins. Specifically, Zn²⁺ binding to disordered regions commonly induces a disorder to order transition and gain of structure or oligomerization. Here we show that simultaneous binding of Zn²⁺ ions has different effects on structured and disordered domains in the same multidomain protein. The centrosomal STIL protein bound Zn²⁺ions via both its structured N-terminal domain (NTD) and disordered central region (IDR). Zn²⁺ binding induced structural rearrangement of the structured NTD but promoted oligomerization of the IDR. We suggest that by binding Zn²⁺ STIL acquires a different conformation, which allows its oligomerization and induces its activity. Sequence alignment of the oligomerization region revealed a new suggested motif, SxKxS/SxHxS/SxLxS, which may participate in STIL oligomerization. Binding of the same metal ion through a disordered and a structured domain in the same protein is a property that may have implications in regulating the protein activity. By doing so, the protein achieves two parallel outcomes: structural changes and oligomerization that can take place together. Our results describe a new important role of the delicate interplay between structure and intrinsic disorder in proteins.

Several bacterial sensory-kinase receptors form clusters on the cell membrane. However, the dynamics of sensory-kinase clustering are largely unclear. Using measurements of fluorescence anisotropy and time-lapse imaging of Escherichia coli cells, we demonstrate that copper ions trigger self-association of BaeS receptors and lead to rapid formation of clusters, which can be reversibly dispersed by a metal chelator. Copper ions did not trigger self-association of other fluorescently tagged sensory kinases, and other divalent metal ions could not elicit self-association of BaeS. The histidine residues in the BaeS periplasmic domain are essential for copper binding in vitro and are important for the copper-induced BaeS responses in vivo. BaeS clustering was triggered also under conditions that directly triggered BaeS-dependent transcriptional responses. Thus, clustering of sensory kinase receptors can be dynamic and context dependent and can be triggered by specific environmental cues.