Molecular Mechanism of STIL Coiled-Coil Domain Oligomerization.

Coiled-coil domains (CCDs) play key roles in regulating both healthy cellular processes and the pathogenesis of various diseases by controlling protein self-association and protein-protein interactions. Here, we probe the mechanism of oligomerization of a peptide representing the CCD of the STIL protein, a tetrameric multi-domain protein that is over-expressed in several cancers and associated with metastatic spread. STIL tetramerization is mediated both by an intrinsically disordered domain (STIL400-700) and a structured CCD (STIL CCD718-749). Disrupting STIL oligomerization via the CCD inhibits its activity in vivo. We describe a comprehensive biophysical and structural characterization of the concentration-dependent oligomerization of STIL CCD peptide. We combine analytical ultracentrifugation, fluorescence and circular dichroism spectroscopy to probe the STIL CCD peptide assembly in solution and determine dissociation constants of both the dimerization, (KD = 8 ± 2 µM) and tetramerization (KD = 68 ± 2 µM) of the WT STIL CCD peptide. The higher-order oligomers result in increased thermal stability and cooperativity of association. We suggest that this complex oligomerization mechanism regulates the activated levels of STIL in the cell and during centriole duplication. In addition, we present X-ray crystal structures for the CCD containing destabilising (L736E) and stabilising (Q729L) mutations, which reveal dimeric and tetrameric antiparallel coiled-coil structures, respectively. Overall, this study offers a basis for understanding the structural molecular biology of the STIL protein, and how it might be targeted to discover anti-cancer reagents.

Amyloid Aggregation Is Potently Slowed Down by Osmolytes Due to Compaction of Partially Folded State.

Amyloid aggregation is a key process in amyloidoses and neurodegenerative diseases. Hydrophobicity is one of the major driving forces for this type of aggregation, as an increase in hydrophobicity generally correlates with aggregation susceptibility and rate. However, most experimental systems in vitro and prediction tools in silico neglect the contribution of protective osmolytes present in the cellular environment. Here, we assessed the role of hydrophobic mutations in amyloid aggregation in the presence of osmolytes. To achieve this goal, we used the model protein human muscle acylphosphatase (mAcP) and mutations to leucine that increased its hydrophobicity without affecting its thermodynamic stability. Osmolytes significantly slowed down the aggregation kinetics of the hydrophobic mutants, with an effect larger than that observed on the wild-type protein. The effect increased as the mutation site was closer to the middle of the protein sequence. We propose that the preferential exclusion of osmolytes from mutation-introduced hydrophobic side-chains quenches the aggregation potential of the ensemble of partially unfolded states of the protein by inducing its compaction and inhibiting its self-assembly with other proteins. Our results suggest that including the effect of the cellular environment in experimental setups and predictive softwares, for both mechanistic studies and drug design, is essential in order to obtain a more complete combination of the driving forces of amyloid aggregation.

A combination of a cell penetrating peptide and a protein translation inhibitor kills metastatic breast cancer cells.

Cell Penetrating Peptides (CPPs) are promising anticancer and antimicrobial drugs. We recently reported that a peptide derived from the human mitochondrial/ER membrane-anchored NEET protein, Nutrient Autophagy Factor 1 (NAF-1; NAF-144-67), selectively permeates and kills human metastatic epithelial breast cancer cells (MDA-MB-231), but not control epithelial cells. As cancer cells alter their phenotype during growth and metastasis, we tested whether NAF-144-67 would also be efficient in killing other human epithelial breast cancer cells that may have a different phenotype. Here we report that NAF-144-67 is efficient in killing BT-549, Hs 578T, MDA-MB-436, and MDA-MB-453 breast cancer cells, but that MDA-MB-157 cells are resistant to it. Upon closer examination, we found that MDA-MB-157 cells display a high content of intracellular vesicles and cellular protrusions, compared to MDA-MB-231 cells, that could protect them from NAF-144-67. Inhibiting the formation of intracellular vesicles and dynamics of cellular protrusions of MDA-MB-157 cells, using a protein translation inhibitor (the antibiotic Cycloheximide), rendered these cells highly susceptible to NAF-144-67, suggesting that under certain conditions, the killing effect of CPPs could be augmented when they are applied in combination with an antibiotic or chemotherapy agent. These findings could prove important for the treatment of metastatic cancers with CPPs and/or treatment combinations that include CPPs.

Visualization of Molecular Permeation into a Multi-compartment Phospholipid Vesicle.

Passive permeation of small molecules into vesicles with multiple compartments is a critical event in many chemical and biological processes. We consider the translocation of the peptide NAF-144-67 labeled with a fluorescent fluorescein dye across membranes of rhodamine-labeled 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) into liposomes with internal vesicles. Time-resolved microscopy revealed a sequential absorbance of the peptide in both the outer and inner micrometer vesicles that developed over a time period of minutes to hours, illustrating the spatial and temporal progress of the permeation. There is minimal perturbation of the membrane structure and no evidence for pore formation. On the basis of molecular dynamics simulations of NAF-144-67, we extended a local defect model to migration processes that include multiple compartments. The model captures the long residence time of the peptide within the membrane and the rate of permeation through the liposome and its internal compartments. Imaging experiments confirm the semi-quantitative description of the permeation of the model by activated diffusion and open the way for studies of more complex systems.

The Oligomerization Domains of the APC Protein Mediate Liquid-Liquid Phase Separation That Is Phosphorylation Controlled.

One of the most important properties of intrinsically disordered proteins is their ability to undergo liquid-liquid phase separation and form droplets. The Adenomatous Polyposis Coli (APC) protein is an IDP that plays a key role in Wnt signaling and mutations in Apc initiate cancer. APC forms droplets via its 20R domains and self-association domain (ASAD) and in the context of Axin. However, the mechanism involved is unknown. Here, we used peptides to study the molecular mechanism and regulation of APC droplet formation. We found that a peptide derived from the ASAD of APC-formed droplets. Peptide array screening showed that the ASAD bound other APC peptides corresponding to the 20R3 and 20R5 domains. We discovered that the 20R3/5 peptides also formed droplets by themselves and mapped specific residues within 20R3/5 that are necessary for droplet formation. When incubated together, the ASAD and 20R3/5 did not form droplets. Thus, the interaction of the ASAD with 20R3 and 20R5 may regulate the droplet formation as a means of regulating different cellular functions. Phosphorylation of 20R3 or 20R5 at specific residues prevented droplet formation of 20R3/5. Our results reveal that phosphorylation and the ability to undergo liquid-liquid phase separation, which are both important properties of intrinsically disordered proteins, are related to each other in APC. Phosphorylation inhibited the liquid-liquid phase separation of APC, acting as an 'on-off' switch for droplet formation. Phosphorylation may thus be a common mechanism regulating LLPS in intrinsically disordered proteins.

Using a Single Peptide to Electrochemically Sense Multiple Kinases.

Kinases are responsible for regulating cellular and physiological processes, and abnormal kinase activity is associated with various diseases. Therefore, kinases are being used as biomarkers for disease and developing methods for their sensing is highly important. Usually more than one kinase is involved in phosphorylating a target protein. However, kinase detection methods usually detect the activity of only one specific kinase. Here we describe an electrochemical kinase sensing tool for the selective detection of two kinases using the same target peptide. We demonstrate the sensing of kinases ERK2 and PKCδ. This is based on a single sensing element, a peptide that contains two distinct phosphorylation sites of these two kinases. Reversibility experiments with alkaline phosphatase and reaction with the electrochemically active ferrocene-labeled ATP showed that the mechanism of sensing is by detecting the enzymatic phosphorylation. Our approach can be further utilized to develop devices for the detection of multiple kinases and can be expanded to other types of enzymes involved in disease.

A peptide-derived strategy for specifically targeting the mitochondria and ER of cancer cells: a new approach in fighting cancer.

An effective anti-cancer therapy should exclusively target cancer cells and trigger in them a broad spectrum of cell death pathways that will prevent avoidance. Here, we present a new approach in cancer therapy that specifically targets the mitochondria and ER of cancer cells. We developed a peptide derived from the flexible and transmembrane domains of the human protein NAF-1/CISD2. This peptide (NAF-144-67) specifically permeates through the plasma membranes of human epithelial breast cancer cells, abolishes their mitochondria and ER, and triggers cell death with characteristics of apoptosis, ferroptosis and necroptosis. In vivo analysis revealed that the peptide significantly decreases tumor growth in mice carrying xenograft human tumors. Computational simulations of cancer vs. normal cell membranes reveal that the specificity of the peptide to cancer cells is due to its selective recognition of their membrane composition. NAF-144-67 represents a promising anti-cancer lead compound that acts via a unique mechanism.

Targeting Protein Interaction Hotspots Using Structured and Disordered Chimeric Peptide Inhibitors.

The main challenge in inhibiting protein-protein interactions (PPI) for therapeutic purposes is designing molecules that bind specifically to the interaction hotspots. Adding to the complexity, such hotspots can be within both structured and disordered interaction interfaces. To address this, we present a strategy for inhibiting the structured and disordered hotspots of interactions using chimeric peptides that contain both structured and disordered parts. The chimeric peptides we developed are comprised of a cyclic structured part and a disordered part, which target both disordered and structured hotspots. We demonstrate our approach by developing peptide inhibitors for the interactions of the antiapoptotic iASPP protein. First, we developed a structured, α-helical stapled peptide inhibitor, derived from the N-terminal domain of MDM2. The peptide bound two hotspots on iASPP at the low micromolar range and had a cytotoxic effect on A2780 cancer cells with a half-maximal inhibitory concentration (IC50) value of 10 ± 1 µM. We then developed chimeric peptides comprising the structured stapled helical peptide and the disordered p53-derived LinkTer peptide that we previously showed to inhibit iASPP by targeting its disordered RT loop. The chimeric peptide targeted both structured and disordered domains in iASPP with higher affinity compared to the individual structured and disordered peptides and caused cancer cell death. Our strategy overcomes the inherent difficulty in inhibiting the interactions of proteins that possess structured and disordered regions. It does so by using chimeric peptides derived from different interaction partners that together target a much wider interface covering both the structured and disordered domains. This paves the way for developing such inhibitors for therapeutic purposes.

Peptide Permeation across a Phosphocholine Membrane: An Atomically Detailed Mechanism Determined through Simulations and Supported by Experimentation.

Cell-penetrating peptides (CPPs) facilitate translocation across biological membranes and are of significant biological and medical interest. Several CPPs can permeate into specific cells and organelles. We examine the incorporation and translocation of a novel anticancer CPP in a dioleoylphosphatidylcholine (DOPC) lipid bilayer membrane. The peptide, NAF-144-67, is a short fragment of a transmembrane protein, consisting of hydrophobic N-terminal and charged C-terminal segments. Experiments using fluorescently labeled NAF-144-67 in ∼100 nm DOPC vesicles and atomically detailed simulations conducted with Milestoning support a model in which a significant barrier for peptide-membrane entry is found at the interface between the aqueous solution and membrane. The initial step is the insertion of the N-terminal segment and the hydrophobic helix into the membrane, passing the hydrophilic head groups. Both experiments and simulations suggest that the free energy difference in the first step of the permeation mechanism in which the hydrophobic helix crosses the phospholipid head groups is -0.4 kcal mol-1 slightly favoring motion into the membrane. Milestoning calculations of the mean first passage time and the committor function underscore the existence of an early polar barrier followed by a diffusive barrierless motion in the lipid tail region. Permeation events are coupled to membrane fluctuations that are examined in detail. Our study opens the way to investigate in atomistic resolution the molecular mechanism, kinetics, and thermodynamics of CPP permeation to diverse membranes.

Electrochemical biosensors based on peptide-kinase interactions at the kinase docking site.

Kinases are important cancer biomarkers and are conventionally detected based on their catalytic activity. Kinases regulate cellular activities by phosphorylation of motif-specific multiple substrate proteins, resulting in a lack of selectivity of activity-based kinase biosensors. We present an alternative approach of sensing kinases based on the interactions of their allosteric docking sites with a specific partner protein. The new approach was demonstrated for the ERK2 kinase and its substrate ELK-1. A peptide derived from ELK-1 was bound to a gold electrode and ERK2 sensing was performed by electrochemical impedance spectroscopy. We performed a detailed analysis of the interaction between the ELK-1 peptide and the kinase on gold surfaces. Atomic force microscopy, variable angle spectroscopic ellipsometry, X-ray Photoelectron Spectroscopy, and polarization modulation IR reflection-absorption spectroscopy analysis of the gold surface revealed the adsorbed layer of the ERK2 on the peptide monolayer. The sensors showed a high level of target selectivity for ERK2 compared to the p38γ kinase and BSA. ERK2 was detected in its cellular concentration range, 0.5-2.0 µM, and the limit of detection was calculated to be 0.35 µM. Using the flexibility of peptide design, our method is generic for developing sensitive and substrate-specific biosensors and other disease-related enzymes based on their interactions.

Kinase Sensing Based on Protein Interactions at the Catalytic Site.

Invited for the cover of this issue is Assaf Friedler, Shlomo Yitzchaik and co-workers at the Hebrew University of Jerusalem and the Academia Sinica. The image depicts a new approach for electrochemical kinase sensing that does not rely on phosphorylation. The kinase binds a peptide layer, which undergoes rearrangement, resulting in the permeation of redox-active species through the layer and electrochemical sensing. Read the full text of the article at 10.1002/chem.202104227.

Kinase Sensing Based on Protein Interactions at the Catalytic Site.

The role kinases play in regulating cellular processes makes them potential biomarkers for detecting the onset and prognosis of various diseases, including many types of cancer. Current kinase biosensors, including electrochemical and radiometric methods, rely on sensing the ATP-dependant enzymatic phosphorylation reaction. Here we introduce a new type of interaction-based electrochemical kinase biosensor that does not require any chemical labelling or modification. The basis for sensing is the interactions between the catalytic site of the kinase and the phosphorylation site of its substrate rather than the phosphorylation reaction. We demonstrated this concept with the ERK2 kinase and its substrate protein HDGF, which is involved in lung cancer. A peptide monolayer derived from the HDGF phosphorylation site was adsorbed onto a gold electrode and was used to sense ERK2 without ATP. The sensitivity of the assay was down to 10 nM of ERK2, corresponding with the range of its cellular concentrations. Surface chemistry analysis confirmed that ERK2 was bound to the HDGF peptide monolayer. This increased the permeability of redox-active species through the monolayer and resulted in ERK2 electrochemical sensing. Since our detection approach is based on protein-protein interactions and not on the enzymatic reaction, it can be further utilized for more selective detection of different types of enzymes.

Osmolytes and crowders regulate aggregation of the cancer-related L106R mutant of the Axin protein.

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 L106R in vitro in the presence of a wide ensemble of cosolutes including polyols, amino acids, betaine, and polyethylene glycol crowders. Except myo-inositol, all polyols decreased RGS L106R aggregation, with carbohydrates exerting the strongest inhibition. Conversely, betaine and polyethylene glycols 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 soluble molecule but the increased crowding of the bound state by macromolecules induces its aggregation at the nanoscale. To our knowledge, this is the first systematic study on the effect of osmolytes and crowders on a process of native-like aggregation involved in pathology, as it sheds light on the contribution of cosolutes to the onset of cancer as a protein misfolding disease and on the relevance of aggregation in the molecular etiology of cancer.

Integrating site-specific peptide reporters and targeted mass spectrometry enables rapid substrate-specific kinase assay at the nanogram cell level.

Dysregulation of phosphorylation-mediated signaling drives the initiation and progression of many diseases. A substrate-specific kinase assay capable of quantifying the altered site-specific phosphorylation of its phenotype-dependent substrates provides better specificity to monitor a disease state. We report a sensitive and rapid substrate-specific kinase assay by integrating site-specific peptide reporter and multiple reaction monitoring (MRM)-MS platform for relative and absolute quantification of substrate-specific kinase activity at the sensitivity of nanomolar kinase and nanogram cell lysate. Using non-small cell lung cancer as a proof-of-concept, three substrate peptides selected from constitutive phosphorylation in tumors (HDGF-S165, RALY-S135, and NRD1-S94) were designed to demonstrate the feasibility. The assay showed good accuracy (<15% nominal deviation) and reproducibility (<15% CV). In PC9 cells, the measured activity for HDGF-S165 was 3.2 ± 0.2 fmol µg-1 min-1, while RALY-S135 and NRD1-S94 showed 4- and 20-fold higher activity at the sensitivity of 25 ng and 5 ng lysate, respectively, suggesting different endogenous kinases for each substrate peptide. Without the conventional shotgun phosphoproteomics workflow, the overall pipeline from cell lysate to MS data acquisition only takes 3 h. The multiplexed analysis revealed differences in the phenotype-dependent substrate phosphorylation profiles across six NSCLC cell lines and suggested a potential association of HDGF-S165 and NRD1-S94 with TKI resistance. With the ease of design, sensitivity, accuracy, and reproducibility, this approach may offer rapid and sensitive assays for targeted quantification of the multiplexed substrate-specific kinase activity of small amounts of sample.

Targeting an Interaction Between Two Disordered Domains by Using a Designed Peptide.

Invited for the cover of this issue is the group of Assaf Friedler at the Hebrew University of Jerusalem. The image depicts the protein-protein interactions reported in this work. Read the full text of the article at 10.1002/chem.202000465.

Multiphosphorylated peptides: importance, synthetic strategies, and applications for studying biological mechanisms.

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.

Targeting an Interaction Between Two Disordered Domains by Using a Designed 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.

Phosphorylation of the WWOX Protein Regulates Its Interaction with p73.

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.

The anti-apoptotic proteins NAF-1 and iASPP interact to drive apoptosis in cancer cells.

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.

Wild-type and cancer-related p53 proteins are preferentially degraded by MDM2 as dimers rather than tetramers.

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.