The MYCN oncoprotein is an RNA-binding accessory factor of the nuclear exosome targeting complex.

The MYCN oncoprotein binds active promoters in a heterodimer with its partner protein MAX. MYCN also interacts with the nuclear exosome, a 3'-5' exoribonuclease complex, suggesting a function in RNA metabolism. Here, we show that MYCN forms stable high-molecular-weight complexes with the exosome and multiple RNA-binding proteins. MYCN binds RNA in vitro and in cells via a conserved sequence termed MYCBoxI. In cells, MYCN associates with thousands of intronic transcripts together with the ZCCHC8 subunit of the nuclear exosome targeting complex and enhances their processing. Perturbing exosome function results in global re-localization of MYCN from promoters to intronic RNAs. On chromatin, MYCN is then replaced by the MNT(MXD6) repressor protein, inhibiting MYCN-dependent transcription. RNA-binding-deficient alleles show that RNA-binding limits MYCN's ability to activate cell growth-related genes but is required for MYCN's ability to promote progression through S phase and enhance the stress resilience of neuroblastoma cells.

MYC Recruits SPT5 to RNA Polymerase II to Promote Processive Transcription Elongation.

The MYC oncoprotein binds to promoter-proximal regions of virtually all transcribed genes and enhances RNA polymerase II (Pol II) function, but its precise mode of action is poorly understood. Using mass spectrometry of both MYC and Pol II complexes, we show here that MYC controls the assembly of Pol II with a small set of transcription elongation factors that includes SPT5, a subunit of the elongation factor DSIF. MYC directly binds SPT5, recruits SPT5 to promoters, and enables the CDK7-dependent transfer of SPT5 onto Pol II. Consistent with known functions of SPT5, MYC is required for fast and processive transcription elongation. Intriguingly, the high levels of MYC that are expressed in tumors sequester SPT5 into non-functional complexes, thereby decreasing the expression of growth-suppressive genes. Altogether, these results argue that MYC controls the productive assembly of processive Pol II elongation complexes and provide insight into how oncogenic levels of MYC permit uncontrolled cellular growth.

Amino-terminally elongated Aß peptides are generated by the secreted metalloprotease ADAMTS4 and deposit in a subset of Alzheimer's disease brains.

AIMS: The aggregation and deposition of amyloid-ß (Aß) peptides in the brain is thought to be the initial driver in the pathogenesis of Alzheimer's disease (AD). Aside from full-length Aß peptides starting with an aspartate residue in position 1, both N-terminally truncated and elongated Aß peptides are produced by various proteases from the amyloid precursor protein (APP) and have been detected in brain tissues and body fluids. Recently, we demonstrated that the particularly abundant N-terminally truncated Aß4-x peptides are generated by ADAMTS4, a secreted metalloprotease that is exclusively expressed in the oligodendrocyte cell population. In this study, we investigated whether ADAMTS4 might also be involved in the generation of N-terminally elongated Aß peptides. METHODS: We used cell-free and cell-based assays in combination with matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF) and electrochemiluminescence sandwich immunoassays to identify and quantify N-terminally elongated Aß peptide variants. Antibodies against these Aß variants were characterised by peptide microarrays and employed for the immunohistochemical analyses of human brain samples. RESULTS: In this study, we discovered additional ADAMTS4 cleavage sites in APP. These were located N-terminal to Asp-(1) in the Aß peptide sequence between residues Glu-(-7) and Ile-(-6) as well as Glu-(-4) and Val-(-3), resulting in the release of N-terminally elongated Aß-6-x and Aß-3-x peptides, of which the latter serve as a component in a promising Aß-based plasma biomarker. Aß-6/-3-40 peptides were detected in supernatants of various cell lines and in the cerebrospinal fluid (CSF), and ADAMTS4 enzyme activity promoted the release of Aß-6/-3-x peptides. Furthermore, by immunohistochemistry, a subset of AD cases displayed evidence of extracellular and vascular localization of N-terminally elongated Aß-6/-3-x peptides. DISCUSSION: The current findings implicate ADAMTS4 in both the pathological process of Aß peptide aggregation and in the early detection of amyloid pathology in AD.

Interaction of YAP with the Myb-MuvB (MMB) complex defines a transcriptional program to promote the proliferation of cardiomyocytes.

The Hippo signalling pathway and its central effector YAP regulate proliferation of cardiomyocytes and growth of the heart. Using genetic models in mice we show that the increased proliferation of embryonal and postnatal cardiomyocytes due to loss of the Hippo-signaling component SAV1 depends on the Myb-MuvB (MMB) complex. Similarly, proliferation of postnatal cardiomyocytes induced by constitutive active YAP requires MMB. Genome studies revealed that YAP and MMB regulate an overlapping set of cell cycle genes in cardiomyocytes. Protein-protein interaction studies in cell lines and with recombinant proteins showed that YAP binds directly to B-MYB, a subunit of MMB, in a manner dependent on the YAP WW domains and a PPXY motif in B-MYB. Disruption of the interaction by overexpression of the YAP binding domain of B-MYB strongly inhibits the proliferation of cardiomyocytes. Our results point to MMB as a critical downstream effector of YAP in the control of cardiomyocyte proliferation.

Gephyrin-binding peptides visualize postsynaptic sites and modulate neurotransmission.

γ-Aminobutyric acid type A and glycine receptors are the major mediators of fast synaptic inhibition in the human central nervous system and are established drug targets. However, all drugs targeting these receptors bind to the extracellular ligand-binding domain of the receptors, which inherently is associated with perturbation of the basic physiological action. Here we pursue a fundamentally different approach, by instead targeting the intracellular receptor-gephyrin interaction. First, we defined the gephyrin peptide-binding consensus sequence, which facilitated the development of gephyrin super-binding peptides and later effective affinity probes for the isolation of native gephyrin. Next, we demonstrated that fluorescent super-binding peptides could be used to directly visualize inhibitory postsynaptic sites for the first time in conventional and super-resolution microscopy. Finally, we demonstrate that the gephyrin super-binding peptides act as acute intracellular modulators of fast synaptic inhibition by modulating receptor clustering, thus being conceptually novel modulators of inhibitory neurotransmission.

Design and synthesis of high-affinity dimeric inhibitors targeting the interactions between gephyrin and inhibitory neurotransmitter receptors.

Gephyrin is the central scaffolding protein for inhibitory neurotransmitter receptors in the brain. Here we describe the development of dimeric peptides that inhibit the interaction between gephyrin and these receptors, a process which is fundamental to numerous synaptic functions and diseases of the brain. We first identified receptor-derived minimal gephyrin-binding peptides that displayed exclusive binding towards native gephyrin from brain lysates. We then designed and synthesized a series of dimeric ligands, which led to a remarkable 1220-fold enhancement of the gephyrin affinity (KD=6.8 nM). In X-ray crystal structures we visualized the simultaneous dimer-to-dimer binding in atomic detail, revealing compound-specific binding modes. Thus, we defined the molecular basis of the affinity-enhancing effect of multivalent gephyrin inhibitors and provide conceptually novel compounds with therapeutic potential, which will allow further elucidation of the gephyrin-receptor interplay.

Inhibition of the YAP-MMB interaction and targeting NEK2 as potential therapeutic strategies for YAP-driven cancers.

YAP activation in cancer is linked to poor outcomes, making it an attractive therapeutic target. Previous research focused on blocking the interaction of YAP with TEAD transcription factors. Here, we took a different approach by disrupting YAP's binding to the transcription factor B-MYB using MY-COMP, a fragment of B-MYB containing the YAP binding domain fused to a nuclear localization signal. MY-COMP induced cell cycle defects, nuclear abnormalities, and polyploidization. In an AKT and YAP-driven liver cancer model, MY-COMP significantly reduced liver tumorigenesis, highlighting the importance of the YAP-B-MYB interaction in tumor development. MY-COMP also perturbed the cell cycle progression of YAP-dependent uveal melanoma cells but not of YAP-independent cutaneous melanoma cell lines. It counteracted YAP-dependent expression of MMB-regulated cell cycle genes, explaining the observed effects. We also identified NIMA-related kinase (NEK2) as a downstream target of YAP and B-MYB, promoting YAP-driven transformation by facilitating centrosome clustering and inhibiting multipolar mitosis.

Peptide Microarrays for Studying Autoantibodies in Neurological Disease.

Antibody-mediated neurological diseases constitute an emerging clinical entity that remains to be fully explored. Recent studies identified autoantibodies that directly confer pathogenicity, and it was shown that in these cases immunotherapies can result in profound positive patient responses. These advances highlight the urgent need for improved means to effectively screen patient samples for novel autoantibodies (aAbs) and their subsequent characterization. Here, we discuss challenges and opportunities for peptide microarrays to contribute to the identification, mapping, and characterization of the underlying monospecific disease-defining binding surfaces. We outline control experiments, workflow modifications and bioinformatic filtering methods that enhance the predictive power of array-based studies. Further, we highlight experimental and computer-based display approaches that have the potential to expand the use of synthetic microarrays over the detection of discontinuous epitopes. Knowledge over the autoantibody epitopes in neurological disease will enhance our understanding of the pathological mechanisms and thereby potentially contribute to novel diagnostic approaches or even innovative antigen-specific treatments that avoid the serious adverse effects seen with currently used immunosuppressive therapies.

Peptide Microarray-Based Protein Interaction Studies Across Affinity Ranges: Enzyme Stalling, Cross-Linking, Depletion, and Neutralization.

While an ever-increasing number of protein-protein interactions were studied by peptide microarrays with great success, array-based investigations of transiently binding proteins, such as HDACs, and precise binding quantification, remained challenging. Here, we present an updated protocol for the preparation and use of peptide microarrays including the necessary adjustments for simple semi-quantitative and precise measurements across affinity ranges. This procedure describes the mass spectrometric controlled preparation of peptide microarrays in µSPOT format, and their application in binding profiling of recombinant, as well as endogenous, native proteins. We further highlight how cross-linking, blocking, and enzyme stalling can be leveraged to enhance sensitivity and describe how in situ on-chip binding neutralization can enhance the predictive value and robustness of the binding readout. Finally, we included examples for the integration of precise biophysical binding readouts that complement the traditional array-based binding assays.

Molecular characterization of AIFM2/FSP1 inhibition by iFSP1-like molecules.

Ferroptosis is a form of cell death characterized by phospholipid peroxidation, where numerous studies have suggested that the induction of ferroptosis is a therapeutic strategy to target therapy refractory cancer entities. Ferroptosis suppressor protein 1 (FSP1), an NAD(P)H-ubiquinone reductase, is a key determinant of ferroptosis vulnerability, and its pharmacological inhibition was shown to strongly sensitize cancer cells to ferroptosis. A first generation of FSP1 inhibitors, exemplified by the small molecule iFSP1, has been reported; however, the molecular mechanisms underlying inhibition have not been characterized in detail. In this study, we explore the species-specific inhibition of iFSP1 on the human isoform to gain insights into its mechanism of action. Using a combination of cellular, biochemical, and computational methods, we establish a critical contribution of a species-specific aromatic architecture that is essential for target engagement. The results described here provide valuable insights for the rational development of second-generation FSP1 inhibitors combined with a tracer for screening the druggable pocket. In addition, we pose a cautionary notice for using iFSP1 in animal models, specifically murine models.

Synapsin autoantibodies during pregnancy are associated with fetal abnormalities.

Anti-neuronal autoantibodies can be transplacentally transferred during pregnancy and may cause detrimental effects on fetal development. It is unclear whether autoantibodies against synapsin-I, one of the most abundant synaptic proteins, are associated with developmental abnormalities in humans. We recruited a cohort of 263 pregnant women and detected serum synapsin-I IgG autoantibodies in 13.3% using cell-based assays. Seropositivity was strongly associated with abnormalities of fetal development including structural defects, intrauterine growth retardation, amniotic fluid disorders and neuropsychiatric developmental diseases in previous children (odds ratios of 3-6.5). Autoantibodies reached the fetal circulation and were mainly of IgG1/IgG3 subclasses. They bound to conformational and linear synapsin-I epitopes, five distinct epitopes were identified using peptide microarrays. The findings indicate that synapsin-I autoantibodies may be clinically useful biomarkers or even directly participate in the disease process of neurodevelopmental disorders, thus being potentially amenable to antibody-targeting interventional strategies in the future.

The residence time of GABA(A)Rs at inhibitory synapses is determined by direct binding of the receptor α1 subunit to gephyrin.

The majority of fast synaptic inhibition in the brain is mediated by benzodiazepine-sensitive α1-subunit-containing GABA type A receptors (GABA(A)Rs); however, our knowledge of the mechanisms neurons use to regulate their synaptic accumulation is rudimentary. Using immunoprecipitation, we demonstrate that GABA(A)Rs and gephyrin are intimately associated at inhibitory synapses in cultured rat neurons. In vitro we reveal that the E-domain of gephyrin directly binds to the α1 subunit with an affinity of ∼20 µm, mediated by residues 360-375 within the intracellular domain of this receptor subunit. Mutating residues 360-375 decreases both the accumulation of α1-containing GABA(A)Rs at gephyrin-positive inhibitory synapses in hippocampal neurons and the amplitude of mIPSCs. We also demonstrate that the affinity of gephyrin for the α1 subunit is modulated by Thr375, a putative phosphorylation site. Mutation of Thr375 to a phosphomimetic, negatively charged amino acid decreases both the affinity of the α1 subunit for gephyrin, and therefore receptor accumulation at synapses, and the amplitude of mIPSCs. Finally, single-particle tracking reveals that gephyrin reduces the diffusion of α1-subunit-containing GABA(A)Rs specifically at inhibitory synapses, thereby increasing their confinement at these structures. Our results suggest that the direct binding of gephyrin to residues 360-375 of the α1 subunit and its modulation are likely to be important determinants for the stabilization of GABA(A)Rs at synaptic sites, thereby modulating the strength of synaptic inhibition.

Gephyrin-mediated γ-aminobutyric acid type A and glycine receptor clustering relies on a common binding site.

Gephyrin is the major protein determinant for the clustering of inhibitory neurotransmitter receptors. Earlier analyses revealed that gephyrin tightly binds to residues 398-410 of the glycine receptor ß subunit (GlyR ß) and, as demonstrated only recently, also interacts with GABA(A) receptors (GABA(A)Rs) containing the α1, α2, and α3 subunits. Here, we dissect the molecular basis underlying the interactions between gephyrin and GABA(A)Rs containing these α-subunits and compare them to the crystal structure of the gephyrin-GlyR ß complex. Biophysical and biochemical assays revealed that, in contrast to its tight interaction with GlyR ß, gephyrin only loosely interacts with GABA(A)R α2, whereas it has an intermediate affinity for the GABA(A)R α1 and α3 subunits. Despite the wide variation in affinities and the low overall sequence homology among the identified receptor subunits, competition assays confirmed the receptor-gephyrin interaction to be a mutually exclusive process. Selected gephyrin point mutants that critically weaken complex formation with GlyR ß also abolished the GABA(A)R α1 and α3 interactions. Additionally, we identified a common binding motif with two conserved aromatic residues that are central for gephyrin binding. Consistent with the biochemical data, mutations of the corresponding residues within the cytoplasmic domain of α2 subunit-containing GABA(A)Rs attenuated clustering of these receptors at postsynaptic sites in hippocampal neurons. Taken together, our experiments provide key insights regarding similarities and differences in the complex formation between gephyrin and GABA(A)Rs compared with GlyRs and, hence, the accumulation of these receptors at postsynaptic sites.

Molecular basis of the γ-aminobutyric acid A receptor α3 subunit interaction with the clustering protein gephyrin.

The multifunctional scaffolding protein gephyrin is a key player in the formation of the postsynaptic scaffold at inhibitory synapses, clustering both inhibitory glycine receptors (GlyRs) and selected GABA(A) receptor (GABA(A)R) subtypes. We report a direct interaction between the GABA(A)R α3 subunit and gephyrin, mapping reciprocal binding sites using mutagenesis, overlay, and yeast two-hybrid assays. This analysis reveals that critical determinants of this interaction are located in the motif FNIVGTTYPI in the GABA(A)R α3 M3-M4 domain and the motif SMDKAFITVL at the N terminus of the gephyrin E domain. GABA(A)R α3 gephyrin binding-site mutants were unable to co-localize with endogenous gephyrin in transfected hippocampal neurons, despite being able to traffic to the cell membrane and form functional benzodiazepine-responsive GABA(A)Rs in recombinant systems. Interestingly, motifs responsible for interactions with GABA(A)R α2, GABA(A)R α3, and collybistin on gephyrin overlap. Curiously, two key residues (Asp-327 and Phe-330) in the GABA(A)R α2 and α3 binding sites on gephyrin also contribute to GlyR ß subunit-E domain interactions. However, isothermal titration calorimetry reveals a 27-fold difference in the interaction strength between GABA(A)R α3 and GlyR ß subunits with gephyrin with dissociation constants of 5.3 µm and 0.2 µm, respectively. Taken together, these observations suggest that clustering of GABA(A)R α2, α3, and GlyRs by gephyrin is mediated by distinct mechanisms at mixed glycinergic/GABAergic synapses.

Multivalent binding kinetics resolved by fluorescence proximity sensing.

Multivalent protein interactors are an attractive modality for probing protein function and exploring novel pharmaceutical strategies. The throughput and precision of state-of-the-art methodologies and workflows for the effective development of multivalent binders is currently limited by surface immobilization, fluorescent labelling and sample consumption. Using the gephyrin protein, the master regulator of the inhibitory synapse, as benchmark, we exemplify the application of Fluorescence proximity sensing (FPS) for the systematic kinetic and thermodynamic optimization of multivalent peptide architectures. High throughput synthesis of +100 peptides with varying combinatorial dimeric, tetrameric, and octameric architectures combined with direct FPS measurements resolved on-rates, off-rates, and dissociation constants with high accuracy and low sample consumption compared to three complementary technologies. The dataset and its machine learning-based analysis deciphered the relationship of specific architectural features and binding kinetics and thereby identified binders with unprecedented protein inhibition capacity; thus, highlighting the value of FPS for the rational engineering of multivalent inhibitors.

Complex regulation of Gephyrin splicing is a determinant of inhibitory postsynaptic diversity.

Gephyrin (GPHN) regulates the clustering of postsynaptic components at inhibitory synapses and is involved in pathophysiology of neuropsychiatric disorders. Here, we uncover an extensive diversity of GPHN transcripts that are tightly controlled by splicing during mouse and human brain development. Proteomic analysis reveals at least a hundred isoforms of GPHN incorporated at inhibitory Glycine and gamma-aminobutyric acid A receptors containing synapses. They exhibit different localization and postsynaptic clustering properties, and altering the expression level of one isoform is sufficient to affect the number, size, and density of inhibitory synapses in cerebellar Purkinje cells. Furthermore, we discovered that splicing defects reported in neuropsychiatric disorders are carried by multiple alternative GPHN transcripts, demonstrating the need for a thorough analysis of the GPHN transcriptome in patients. Overall, we show that alternative splicing of GPHN is an important genetic variation to consider in neurological diseases and a determinant of the diversity of postsynaptic inhibitory synapses.

A bead-based GPCR phosphorylation immunoassay for high-throughput ligand profiling and GRK inhibitor screening.

Analysis of agonist-driven phosphorylation of G protein-coupled receptors (GPCRs) can provide valuable insights into the receptor activation state and ligand pharmacology. However, to date, assessment of GPCR phosphorylation using high-throughput applications has been challenging. We have developed and validated a bead-based immunoassay for the quantitative assessment of agonist-induced GPCR phosphorylation that can be performed entirely in multiwell cell culture plates. The assay involves immunoprecipitation of affinity-tagged receptors using magnetic beads followed by protein detection using phosphorylation state-specific and phosphorylation state-independent anti-GPCR antibodies. As proof of concept, five prototypical GPCRs (MOP, C5a1, D1, SST2, CB2) were treated with different agonizts and antagonists, and concentration-response curves were generated. We then extended our approach to establish selective cellular GPCR kinase (GRK) inhibitor assays, which led to the rapid identification of a selective GRK5/6 inhibitor (LDC8988) and a highly potent pan-GRK inhibitor (LDC9728). In conclusion, this versatile GPCR phosphorylation assay can be used extensively for ligand profiling and inhibitor screening.

Expanding GABAAR pharmacology via receptor-associated proteins.

Drugs directly targeting γ-aminobutyric acid type A receptors (GABAARs), the major mediators of fast synaptic inhibition, contribute significantly to today's neuropharmacology. Emerging evidence establishes intracellularly GABAAR-associated proteins as the central players in determining cellular and subcellular GABAergic input sites, thereby providing pharmacological opportunities to affect distinct receptor populations and address discrete neuronal dysfunctions. Here, we report on recently studied GABAAR-associated proteins and highlight challenges and newly available methods for their functional and physical mapping. We anticipate these efforts to contribute to decipher the complexity of GABAergic signalling in the brain and eventually enable therapeutic avenues for, so far, untreatable neuronal disorders and diseases.

Low-cost synthesis of peptide libraries and their use for binding studies via temperature-related intensity change.

Protein-peptide interactions are involved in many fundamental cellular functions and constitute promising drug targets. Here, we provide a detailed protocol for the cost-effective preparation of a cellulose-based solid support for synthesis of nanoscale to micromolar-scale peptide libraries. Their subsequent use for high-throughput protein interaction screening as well as affinity determination in solution provides binding data for thousands of unique peptides with a turnover of 1 to 2 weeks, thereby facilitating in vitro assessment and development of high-affinity binders. For complete details on the use and execution of this protocol, please refer to Schulte et al., (2020).

Conformational Plasticity of Hepatitis B Core Protein Spikes Promotes Peptide Binding Independent of the Secretion Phenotype.

Hepatitis B virus is a major human pathogen, which forms enveloped virus particles. During viral maturation, membrane-bound hepatitis B surface proteins package hepatitis B core protein capsids. This process is intercepted by certain peptides with an "LLGRMKG" motif that binds to the capsids at the tips of dimeric spikes. With microcalorimetry, electron cryo microscopy and peptide microarray-based screens, we have characterized the structural and thermodynamic properties of peptide binding to hepatitis B core protein capsids with different secretion phenotypes. The peptide "GSLLGRMKGA" binds weakly to hepatitis B core protein capsids and mutant capsids with a premature (F97L) or low-secretion phenotype (L60V and P5T). With electron cryo microscopy, we provide novel structures for L60V and P5T and demonstrate that binding occurs at the tips of the spikes at the dimer interface, splaying the helices apart independent of the secretion phenotype. Peptide array screening identifies "SLLGRM" as the core binding motif. This shortened motif binds only to one of the two spikes in the asymmetric unit of the capsid and induces a much smaller conformational change. Altogether, these comprehensive studies suggest that the tips of the spikes act as an autonomous binding platform that is unaffected by mutations that affect secretion phenotypes.