An aggregation inhibitor specific to oligomeric intermediates of Aß42 derived from phage display libraries of stable, small proteins.

The self-assembly of amyloid ß peptide (Aß) to fibrillar and oligomeric aggregates is linked to Alzheimer's disease. Aß binders may serve as inhibitors of aggregation to prevent the generation of neurotoxic species and for the detection of Aß species. A particular challenge involves finding binders to on-pathway oligomers given their transient nature. Here we construct two phage­display libraries built on the highly inert and stable protein scaffold S100G, one containing a six-residue variable surface patch and one harboring a seven-residue variable loop insertion. Monomers and fibrils of Aß40 and Aß42 were separately coupled to silica nanoparticles, using a coupling strategy leading to the presence of oligomers on the monomer beads, and they were used in three rounds of affinity selection. Next-generation sequencing revealed sequence clusters and candidate binding proteins (SXkmers). Two SXkmers were expressed as soluble proteins and tested in terms of aggregation inhibition via thioflavin T fluorescence. We identified an SXkmer with loop­insertion YLTIRLM as an inhibitor of the secondary nucleation of Aß42 and binding analyses using surface plasmon resonance technology, Förster resonance energy transfer, and microfluidics diffusional sizing imply an interaction with intermediate oligomeric species. A linear peptide with the YLTIRLM sequence was found inhibitory but at a lower potency than the more constrained SXkmer loop. We identified an SXkmer with side-patch VI-WI-DD as an inhibitor of Aß40 aggregation. Remarkably, our data imply that SXkmer-YLTIRLM blocks secondary nucleation through an interaction with oligomeric intermediates in solution or at the fibril surface, which is a unique inhibitory mechanism for a library-derived inhibitor.

A chromatin-independent role of Polycomb-like 1 to stabilize p53 and promote cellular quiescence.

Polycomb-like proteins 1-3 (PCL1-3) are substoichiometric components of the Polycomb-repressive complex 2 (PRC2) that are essential for association of the complex with chromatin. However, it remains unclear why three proteins with such apparent functional redundancy exist in mammals. Here we characterize their divergent roles in both positively and negatively regulating cellular proliferation. We show that while PCL2 and PCL3 are E2F-regulated genes expressed in proliferating cells, PCL1 is a p53 target gene predominantly expressed in quiescent cells. Ectopic expression of any PCL protein recruits PRC2 to repress the INK4A gene; however, only PCL2 and PCL3 confer an INK4A-dependent proliferative advantage. Remarkably, PCL1 has evolved a PRC2- and chromatin-independent function to negatively regulate proliferation. We show that PCL1 binds to and stabilizes p53 to induce cellular quiescence. Moreover, depletion of PCL1 phenocopies the defects in maintaining cellular quiescence associated with p53 loss. This newly evolved function is achieved by the binding of the PCL1 N-terminal PHD domain to the C-terminal domain of p53 through two unique serine residues, which were acquired during recent vertebrate evolution. This study illustrates the functional bifurcation of PCL proteins, which act in both a chromatin-dependent and a chromatin-independent manner to regulate the INK4A and p53 pathways.

Resolving the Interactome of the Human Macrophage Immunometabolism Regulator (MACIR) with Enhanced Membrane Protein Preparation and Affinity Proteomics.

Expression of the macrophage immunometabolism regulator gene (MACIR) is associated with severity of autoimmune disease pathology and with the regulation of macrophage biology through unknown mechanisms. The encoded 206 amino acid protein lacks homology to any characterized protein sequence and is a disordered protein according to structure prediction algorithms. To identify interactions of MACIR with proteins from all subcellular compartments, a membrane solubilization buffer is employed, that together with a high affinity EF hand based pull down method, increases the resolution of quantitative mass spectrometry analysis with significant enrichment of interactions from membrane bound nuclear and mitochondrial compartments compared to samples prepared with radioimmunoprecipitation assay buffer. A total of 63 significant interacting proteins are identified and interaction with the nuclear transport receptor TNPO1 and the trafficking proteins UNC119 homolog A and B are validated by immunoprecipitation. Mutational analysis in two candidate nuclear localization signal motifs in the MACIR amino acid sequence shows the interaction with TNPO1 is likely via a non-classical proline/tyrosine-nuclear localization signal motif (aa98-117). It is shown that employing a highly specific and high affinity pull down method that performs efficiently in this glycerol and detergent rich buffer is a powerful approach for the analysis of uncharacterized protein interactomes.

Identification of α-helix 4 (α4) of Rab11a as a novel Rab11-binding domain (RBD): Interaction of Rab11a with the Prostacyclin Receptor.

The cellular trafficking of numerous G protein-coupled receptors (GPCRs) is known to be regulated by Rab proteins that involves a direct protein:protein interaction between the receptor and the GTPase. In the case of the human prostacyclin receptor (hIP), it undergoes agonist-induced internalization and subsequent Rab11a-dependent recyclization involving an interaction between a Rab11-binding domain (RBD) localized within its carboxyl-tail domain with Rab11a. However, the GPCR-interacting domain on Rab11a itself is unknown. Hence, we sought to identify the region within Rab11a that mediates its interaction with the RBD of the hIP. The α4 helix region of Rab11 was identified as a novel binding domain for the hIP, a site entirely distinct from the Switch I/Switch II -regions that act as specific binding domain for most other Rab and Ras-like GTPase interactants. Specifically, Glu138 within α4 helix of Rab11a appears to contact with key residues (e.g. Lys304) within the RBD of the hIP, where such contacts differ depending on the agonist-activated versus -inactive status of the hIP. Through mutational studies, supported by in silico homology modelling of the inactive and active hIP:Rab11a complexes, a mechanism is proposed to explain both the constitutive and agonist-induced binding of Rab11a to regulate intracellular trafficking of the hIP. Collectively, these studies are not only the first to identify α4 helix of Rab11a as a protein binding domain on the GTPase but also reveal novel mechanistic insights into the intracellular trafficking of the hIP, and potentially of other members of the GPCR superfamily, involving Rab11-dependent mechanisms.

Simplifying G Protein-Coupled Receptor Isolation with a Calcium-Dependent Fragment Complementation Affinity System.

The process of isolating recombinant G protein-coupled receptors from membrane preparations is challenging because the process requires solubilization in detergent micelles and multistep affinity chromatography protocols. Solubilization buffers contain high concentrations of salts, detergents, and glycerol that create stringent conditions necessary to stabilize the receptor but in which affinity chromatography resins perform poorly, and these resins also require the addition of eluting agents that complicate downstream assays. To simplify this process we have developed a high affinity fragment complementation molecular switch as a highly specific system for receptor capture in solubilization buffer with a calcium chelation-based elution step releasing functional protein in a simple buffer. Here we describe in detail the design, methodology, interpretation, and limitations of this novel affinity chromatography system in the isolation and purification of the cannabinoid G protein-coupled receptor CB2, in comparison with commercially available systems. This powerful tool may be applied to any recombinant membrane bound protein and can be further optimized to enhance the yield and purity of the most challenging protein targets for study.

Ligand-directed targeting of lymphatic vessels uncovers mechanistic insights in melanoma metastasis.

Metastasis is the most lethal step of cancer progression in patients with invasive melanoma. In most human cancers, including melanoma, tumor dissemination through the lymphatic vasculature provides a major route for tumor metastasis. Unfortunately, molecular mechanisms that facilitate interactions between melanoma cells and lymphatic vessels are unknown. Here, we developed an unbiased approach based on molecular mimicry to identify specific receptors that mediate lymphatic endothelial-melanoma cell interactions and metastasis. By screening combinatorial peptide libraries directly on afferent lymphatic vessels resected from melanoma patients during sentinel lymphatic mapping and lymph node biopsies, we identified a significant cohort of melanoma and lymphatic surface binding peptide sequences. The screening approach was designed so that lymphatic endothelium binding peptides mimic cell surface proteins on tumor cells. Therefore, relevant metastasis and lymphatic markers were biochemically identified, and a comprehensive molecular profile of the lymphatic endothelium during melanoma metastasis was generated. Our results identified expression of the phosphatase 2 regulatory subunit A, α-isoform (PPP2R1A) on the cell surfaces of both melanoma cells and lymphatic endothelial cells. Validation experiments showed that PPP2R1A is expressed on the cell surfaces of both melanoma and lymphatic endothelial cells in vitro as well as independent melanoma patient samples. More importantly, PPP2R1A-PPP2R1A homodimers occur at the cellular level to mediate cell-cell interactions at the lymphatic-tumor interface. Our results revealed that PPP2R1A is a new biomarker for melanoma metastasis and show, for the first time to our knowledge, an active interaction between the lymphatic vasculature and melanoma cells during tumor progression.

A Microfluidic Platform for Real-Time Detection and Quantification of Protein-Ligand Interactions.

The key steps in cellular signaling and regulatory pathways rely on reversible noncovalent protein-ligand binding, yet the equilibrium parameters for such events remain challenging to characterize and quantify in solution. Here, we demonstrate a microfluidic platform for the detection of protein-ligand interactions with an assay time on the second timescale and without the requirement for immobilization or the presence of a highly viscous matrix. Using this approach, we obtain absolute values for the electrophoretic mobilities characterizing solvated proteins and demonstrate quantitative comparison of results obtained under different solution conditions. We apply this strategy to characterize the interaction between calmodulin and creatine kinase, which we identify as a novel calmodulin target. Moreover, we explore the differential calcium ion dependence of calmodulin ligand-binding affinities, a system at the focal point of calcium-mediated cellular signaling pathways. We further explore the effect of calmodulin on creatine kinase activity and show that it is increased by the interaction between the two proteins. These findings demonstrate the potential of quantitative microfluidic techniques to characterize binding equilibria between biomolecules under native solution conditions.

Virtual screening using combinatorial cyclic peptide libraries reveals protein interfaces readily targetable by cyclic peptides.

Protein-protein and protein-peptide interactions are responsible for the vast majority of biological functions in vivo, but targeting these interactions with small molecules has historically been difficult. What is required are efficient combined computational and experimental screening methods to choose among a number of potential protein interfaces worthy of targeting lead macrocyclic compounds for further investigation. To achieve this, we have generated combinatorial 3D virtual libraries of short disulfide-bonded peptides and compared them to pharmacophore models of important protein-protein and protein-peptide structures, including short linear motifs (SLiMs), protein-binding peptides, and turn structures at protein-protein interfaces, built from 3D models available in the Protein Data Bank. We prepared a total of 372 reference pharmacophores, which were matched against 108,659 multiconformer cyclic peptides. After normalization to exclude nonspecific cyclic peptides, the top hits notably are enriched for mimetics of turn structures, including a turn at the interaction surface of human α thrombin, and also feature several protein-binding peptides. The top cyclic peptide hits also cover the critical "hot spot" interaction sites predicted from the interaction crystal structure. We have validated our method by testing cyclic peptides predicted to inhibit thrombin, a key protein in the blood coagulation pathway of important therapeutic interest, identifying a cyclic peptide inhibitor with lead-like activity. We conclude that protein interfaces most readily targetable by cyclic peptides and related macrocyclic drugs may be identified computationally among a set of candidate interfaces, accelerating the choice of interfaces against which lead compounds may be screened.

Vascular ligand-receptor mapping by direct combinatorial selection in cancer patients.

Molecules differentially expressed in blood vessels among organs or between damaged and normal tissues, are attractive therapy targets; however, their identification within the human vasculature is challenging. Here we screened a peptide library in cancer patients to uncover ligand-receptors common or specific to certain vascular beds. Surveying ~2.35 x 10(6) motifs recovered from biopsies yielded a nonrandom distribution, indicating that systemic tissue targeting is feasible. High-throughput analysis by similarity search, protein arrays, and affinity chromatography revealed four native ligand-receptors, three of which were previously unrecognized. Two are shared among multiple tissues (integrin α4/annexin A4 and cathepsin B/apolipoprotein E3) and the other two have a restricted and specific distribution in normal tissue (prohibitin/annexin A2 in white adipose tissue) or cancer (RAGE/leukocyte proteinase-3 in bone metastases). These findings provide vascular molecular markers for biotechnology and medical applications.

Complement factor H is an ICOS ligand modulating Tregs in the glioma microenvironment.

The survival rate of glioma patients has not significantly increased in recent years despite aggressive treatment and advances in immunotherapy. The limited response to treatments is partially attributed to the immunosuppressive tumor microenvironment, where regulatory T cells (Tregs) play a pivotal role in immunological tolerance. In this study, we investigated the impact of complement factor H (FH) on Tregs within the glioma microenvironment and found that FH is an ICOS ligand. The binding of FH to this immune checkpoint molecule promoted the survival and function of Tregs and induced the secretion of TGF-beta (TGF-ß) and IL-10, while also suppressing T-cell proliferation. We further demonstrated that cancer cells in human and mouse gliomas directly produce FH. Database investigations revealed that upregulation of FH expression was associated with the presence of Tregs and correlated with worse prognosis for glioma patients. We confirmed the effect of FH on glioma development in a mouse model, where FH knockdown was associated with decrease in number of ICOS+ Tregs and demonstrated a tendency of prolonged survival (p=0.064). Since the accumulation of Tregs represents a promising prognostic and therapeutic target, evaluating FH expression should be considered when assessing the effectiveness of and resistance to immunotherapies against glioma.

Integrated protein array screening and high throughput validation of 70 novel neural calmodulin-binding proteins.

Calmodulin is an essential regulator of intracellular processes in response to extracellular stimuli mediated by a rise in Ca(2+) ion concentration. To profile protein-protein interactions of calmodulin in human brain, we probed a high content human protein array with fluorophore-labeled calmodulin in the presence of Ca(2+). This protein array contains 37,200 redundant proteins, incorporating over 10,000 unique human neural proteins from a human brain cDNA library. We designed a screen to find high affinity (K(D) < or = 1 microm) binding partners of calmodulin and identified 76 human proteins from all intracellular compartments of which 72 are novel. We measured the binding kinetics of 74 targets with calmodulin using a high throughput surface plasmon resonance assay. Most of the novel calmodulin-target complexes identified have low dissociation rates (k(off) < or = 10(-3) s(-1)) and high affinity (K(D)

Conversion of mammalian cell culture media waste to microbial fermentation feed efficiently supports production of recombinant protein by Escherichia coli.

Deriving new value from waste streams through secondary processes is a central aim of the circular bioeconomy. In this study we investigate whether chemically defined spent media (CDSM) waste from cell culture bioprocess can be recycled and used as a feed in secondary microbial fermentation to produce new recombinant protein products. Our results show that CDSM supplemented with 2% glycerol supported a specific growth rate of E. coli cultures equivalent to that achieved using a nutritionally rich microbiological media (LB). The titre of recombinant protein produced following induction in a 4-hour expression screen was approximately equivalent in the CDSM fed cultures to that of baseline, and this was maintained in a 16-hr preparative fermentation. To understand the protein production achieved in CDSM fed culture we performed a quantitative analysis of proteome changes in the E. coli using mass spectrometry. This analysis revealed significant upregulation of protein synthesis machinery enzymes and significant downregulation of carbohydrate metabolism enzymes. We conclude that spent cell culture media, which represents 100s of millions of litres of waste generated by the bioprocessing industry annually, may be valorized as a feed resource for the production of recombinant proteins in secondary microbial fermentations. Data is available via ProteomeXchange with identifier PXD026884.

Single Step Purification of Glycogen Synthase Kinase Isoforms from Small Scale Transient Expression in HEK293 Cells with a Calcium-Dependent Fragment Complementation System.

Purification of proteins for the biophysical analysis of protein interactions occurring in human cells can benefit from methods that facilitate the capture of small amounts of natively processed protein obtained using transient mammalian expression systems. We have used a novel calcium-dependent fragment complementation-based affinity method to effectively purify full length glycogen synthase kinase 3 (GSK3) α and ß isoforms to study their interaction with amyloid ß peptide (Aß42). Using these proteins, purified from 1 mg of total cell lysate, we measured an apparent KD of ≤100 pM between GSK3α/ß and immobilized Aß42 with surface plasmon resonance technology. This approach can be used to retrieve useful quantities of protein for biophysical experiments with small scale mammalian cell culture.

Complement Component C3 Is Highly Expressed in Human Pancreatic Islets and Prevents ß Cell Death via ATG16L1 Interaction and Autophagy Regulation.

We show here that human pancreatic islets highly express C3, which is both secreted and present in the cytosol. Within isolated human islets, C3 expression correlates with type 2 diabetes (T2D) donor status, HbA1c, and inflammation. Islet C3 expression is also upregulated in several rodent diabetes models. C3 interacts with ATG16L1, which is essential for autophagy. Autophagy relieves cellular stresses faced by ß cells during T2D and maintains cellular homeostasis. C3 knockout in clonal ß cells impaired autophagy and led to increased apoptosis after exposure of cells to palmitic acid and IAPP. In the absence of C3, autophagosomes do not undergo fusion with lysosomes. Thus, C3 may be upregulated in islets during T2D as a cytoprotective factor against ß cell dysfunction caused by impaired autophagy. Therefore, we revealed a previously undescribed intracellular function for C3, connecting the complement system directly to autophagy, with a broad potential importance in other diseases and cell types.

Direct High Affinity Interaction between Aß42 and GSK3α Stimulates Hyperphosphorylation of Tau. A New Molecular Link in Alzheimer's Disease?

Amyloid ß peptide (Aß42) assemblies are considered central to the development of Alzheimer's disease, but the mechanism of this toxicity remains unresolved. We screened protein microarrays with on-pathway oligomeric Aß42 to identify candidate proteins interacting with toxic Aß42 species. Samples prepared from Alexa546-Aß42 and Aß42 monomers at 1:5 molar ratio were incubated with the array during a time window of the amyloid fibril formation reaction during which the maximum number of transient oligomers exist in the reaction flux. A specific interaction was detected between Aß42 and glycogen synthase kinase 3α (GSK3α), a kinase previously implicated in the disease pathology. This interaction was validated with anti-GSK3α immunoprecipitation assays in neuronal cell lysates. Confocal microscopy studies further identified colocalization of Aß42 and GSK3α in neurites of mature primary mouse neurons. A high binding affinity (KD = 1 nM) was measured between Alexa488-Aß42 and GSK3α in solution using thermophoresis. An even lower apparent KD was estimated between GSK3α and dextran-immobilized Aß42 in surface plasmon resonance experiments. Parallel experiments with GSK3ß also identified colocalization and high affinity binding to this isoform. GSK3α-mediated hyperphosphorylation of the protein tau was found to be stimulated by Aß42 in in vitro phosphorylation assays and identified a functional relationship between the proteins. We uncover a direct and functional molecular link between Aß42 and GSK3α, which opens an important avenue toward understanding the mechanism of Aß42-mediated neuronal toxicity in Alzheimer's disease.

Sorcin links calcium signaling to vesicle trafficking, regulates Polo-like kinase 1 and is necessary for mitosis.

Sorcin, a protein overexpressed in many multi-drug resistant cancers, dynamically localizes to distinct subcellular sites in 3T3-L1 fibroblasts during cell-cycle progression. During interphase sorcin is in the nucleus, in the plasma membrane, in endoplasmic reticulum (ER) cisternae, and in ER-derived vesicles localized along the microtubules. These vesicles are positive to RyR, SERCA, calreticulin and Rab10. At the beginning of mitosis, sorcin-containing vesicles associate with the mitotic spindle, and during telophase are concentrated in the cleavage furrow and, subsequently, in the midbody. Sorcin regulates dimensions and calcium load of the ER vesicles by inhibiting RYR and activating SERCA. Analysis of sorcin interactome reveals calcium-dependent interactions with many proteins, including Polo-like kinase 1 (PLK1), Aurora A and Aurora B kinases. Sorcin interacts physically with PLK1, is phosphorylated by PLK1 and induces PLK1 autophosphorylation, thereby regulating kinase activity. Knockdown of sorcin results in major defects in mitosis and cytokinesis, increase in the number of rounded polynucleated cells, blockage of cell progression in G2/M, apoptosis and cell death. Sorcin regulates calcium homeostasis and is necessary for the activation of mitosis and cytokinesis.

CX3CL1 is up-regulated in the rat hippocampus during memory-associated synaptic plasticity.

Several cytokines and chemokines are now known to play normal physiological roles in the brain where they act as key regulators of communication between neurons, glia, and microglia. In particular, cytokines and chemokines can affect cardinal cellular and molecular processes of hippocampal-dependent long-term memory consolidation including synaptic plasticity, synaptic scaling and neurogenesis. The chemokine, CX3CL1 (fractalkine), has been shown to modulate synaptic transmission and long-term potentiation (LTP) in the CA1 pyramidal cell layer of the hippocampus. Here, we confirm widespread expression of CX3CL1 on mature neurons in the adult rat hippocampus. We report an up-regulation in CX3CL1 protein expression in the CA1, CA3 and dentate gyrus (DG) of the rat hippocampus 2 h after spatial learning in the water maze task. Moreover, the same temporal increase in CX3CL1 was evident following LTP-inducing theta-burst stimulation in the DG. At physiologically relevant concentrations, CX3CL1 inhibited LTP maintenance in the DG. This attenuation in dentate LTP was lost in the presence of GABAA receptor/chloride channel antagonism. CX3CL1 also had opposing actions on glutamate-mediated rise in intracellular calcium in hippocampal organotypic slice cultures in the presence and absence of GABAA receptor/chloride channel blockade. Using primary dissociated hippocampal cultures, we established that CX3CL1 reduces glutamate-mediated intracellular calcium rises in both neurons and glia in a dose dependent manner. In conclusion, CX3CL1 is up-regulated in the hippocampus during a brief temporal window following spatial learning the purpose of which may be to regulate glutamate-mediated neurotransmission tone. Our data supports a possible role for this chemokine in the protective plasticity process of synaptic scaling.

CHD5 is required for neurogenesis and has a dual role in facilitating gene expression and polycomb gene repression.

The chromatin remodeler CHD5 is expressed in neural tissue and is frequently deleted in aggressive neuroblastoma. Very little is known about the function of CHD5 in the nervous system or its mechanism of action. Here we report that depletion of Chd5 in the developing neocortex blocks neuronal differentiation and leads to an accumulation of undifferentiated progenitors. CHD5 binds a large cohort of genes and is required for facilitating the activation of neuronal genes. It also binds a cohort of Polycomb targets and is required for the maintenance of H3K27me3 on these genes. Interestingly, the chromodomains of CHD5 directly bind H3K27me3 and are required for neuronal differentiation. In the absence of CHD5, a subgroup of Polycomb-repressed genes becomes aberrantly expressed. These findings provide insights into the regulatory role of CHD5 during neurogenesis and suggest how inactivation of this candidate tumor suppressor might contribute to neuroblastoma.

Epitope presentation is an important determinant of the utility of antigens identified from protein arrays in the development of autoantibody diagnostic assays.

Autoantibodies represent an attractive biomarker for diagnostic assays principally due to the stability of immunoglobulin in patient serum facilitating measurement with conventional assays. Immune responses to tumorigenesis may facilitate detection of ovarian cancer in the early stages of the disease with identification of a panel of tumour specific autoantibodies. Despite the reporting of many tumour associated autoantibodies using arrays of tumour antigens, this has not led to the advance in diagnostic capability as rapidly as was initially expected. Here we examine the potential diagnostic utility of candidate autoantibody biomarkers identified via screening of serum samples on a high content human protein array from a unique cohort of early stage and late stage ovarian cancer patients. We analyse the performance of autoantibodies to the tumour suppressor protein p53 and the novel autoantigens alpha adducin and endosulfine alpha identified in our array screen. Each antigen has different performance characteristics using conventional ELISA format and Western blot immunoassay. The high attrition rate of promising autoantigens identified by array screening can in part be explained by the presentation of the epitope of the antigen in the subsequent method of validation and this study provides directions on maximising the potential of candidate biomarkers. This article is part of a Special Issue entitled: Translational Proteomics.

Polycomb PHF19 binds H3K36me3 and recruits PRC2 and demethylase NO66 to embryonic stem cell genes during differentiation.

Polycomb group proteins are repressive chromatin modifiers with essential roles in metazoan development, cellular differentiation and cell fate maintenance. How Polycomb proteins access active chromatin to confer transcriptional silencing during lineage transitions remains unclear. Here we show that the Polycomb repressive complex 2 (PRC2) component PHF19 binds trimethylated histone H3 Lys36 (H3K36me3), a mark of active chromatin, via its Tudor domain. PHF19 associates with the H3K36me3 demethylase NO66, and it is required to recruit the PRC2 complex and NO66 to stem cell genes during differentiation, leading to PRC2-mediated trimethylation of histone H3 Lys27 (H3K27), loss of H3K36me3 and transcriptional silencing. We propose a model whereby PHF19 functions during mouse embryonic stem cell differentiation to transiently bind the H3K36me3 mark via its Tudor domain, forming essential contact points that allow recruitment of PRC2 and H3K36me3 demethylase activity to active gene loci during their transition to a Polycomb-repressed state.