Systematic analysis of alternative exon-dependent interactome remodeling reveals multitasking functions of gene regulatory factors.

Alternative splicing significantly expands biological complexity, particularly in the vertebrate nervous system. Increasing evidence indicates that developmental and tissue-dependent alternative exons often control protein-protein interactions; yet, only a minor fraction of these events have been characterized. Using affinity purification-mass spectrometry (AP-MS), we show that approximately 60% of analyzed neural-differential exons in proteins previously implicated in transcriptional regulation result in the gain or loss of interaction partners, which in some cases form unexpected links with coupled processes. Notably, a neural exon in Chtop regulates its interaction with the Prmt1 methyltransferase and DExD-Box helicases Ddx39b/a, affecting its methylation and activity in promoting RNA export. Additionally, a neural exon in Sap30bp affects interactions with RNA processing factors, modulating a critical function of Sap30bp in promoting the splicing of <100 nt "mini-introns" that control nuclear RNA levels. AP-MS is thus a powerful approach for elucidating the multifaceted functions of proteins imparted by context-dependent alternative exons.

A central chaperone-like role for 14-3-3 proteins in human cells.

14-3-3 proteins are highly conserved regulatory proteins that interact with hundreds of structurally diverse clients and act as central hubs of signaling networks. However, how 14-3-3 paralogs differ in specificity and how they regulate client protein function are not known for most clients. Here, we map the interactomes of all human 14-3-3 paralogs and systematically characterize the effect of disrupting these interactions on client localization. The loss of 14-3-3 binding leads to the coalescence of a large fraction of clients into discrete foci in a client-specific manner, suggesting a central chaperone-like function for 14-3-3 proteins. Congruently, the engraftment of 14-3-3 binding motifs to nonclients can suppress their aggregation or phase separation. Finally, we show that 14-3-3s negatively regulate the localization of the RNA-binding protein SAMD4A to cytoplasmic granules and inhibit its activity as a translational repressor. Our work suggests that 14-3-3s have a more prominent role as chaperone-like molecules than previously thought.

A quantitative chaperone interaction network reveals the architecture of cellular protein homeostasis pathways.

Chaperones are abundant cellular proteins that promote the folding and function of their substrate proteins (clients). In vivo, chaperones also associate with a large and diverse set of cofactors (cochaperones) that regulate their specificity and function. However, how these cochaperones regulate protein folding and whether they have chaperone-independent biological functions is largely unknown. We combined mass spectrometry and quantitative high-throughput LUMIER assays to systematically characterize the chaperone-cochaperone-client interaction network in human cells. We uncover hundreds of chaperone clients, delineate their participation in specific cochaperone complexes, and establish a surprisingly distinct network of protein-protein interactions for cochaperones. As a salient example of the power of such analysis, we establish that NUDC family cochaperones specifically associate with structurally related but evolutionarily distinct ß-propeller folds. We provide a framework for deciphering the proteostasis network and its regulation in development and disease and expand the use of chaperones as sensors for drug-target engagement.

Comprehensive interactome profiling of the human Hsp70 network highlights functional differentiation of J domains.

Hsp70s comprise a deeply conserved chaperone family that has a central role in maintaining protein homeostasis. In humans, Hsp70 client specificity is provided by 49 different co-factors known as J domain proteins (JDPs). However, the cellular function and client specificity of JDPs have largely remained elusive. We have combined affinity purification-mass spectrometry (AP-MS) and proximity-dependent biotinylation (BioID) to characterize the interactome of all human JDPs and Hsp70s. The resulting network suggests specific functions for many uncharacterized JDPs, and we establish a role of conserved JDPs DNAJC9 and DNAJC27 in histone chaperoning and ciliogenesis, respectively. Unexpectedly, we find that the J domain of DNAJC27 but not of other JDPs can fully replace the function of endogenous DNAJC27, suggesting a previously unappreciated role for J domains themselves in JDP specificity. More broadly, our work expands the role of the Hsp70-regulated proteostasis network and provides a platform for further discovery of JDP-dependent functions.

Interactomes of Glycogen Synthase Kinase-3 Isoforms.

Functional differentiation of the two isoforms of the protein-serine/threonine kinase, glycogen synthase kinase-3 (GSK-3), is an unsettled area of research. The isoforms are highly similar in structure and are largely redundant, though there is also evidence for specific roles. Identification of isoform-specific protein interactors may elucidate the differences in function and provide insight into isoform-selective regulation. We therefore sought to identify novel GSK-3 interaction partners and to examine differences in the interactomes of the two isoforms using both affinity purification and proximity-dependent biotinylation (BioID) mass spectrometry methods. While the interactomes of the two isomers are highly similar in HEK293 cells, BioID in HeLa cells yielded a variety of preys that are preferentially associated with one of the two isoforms. DCP1B, which favored GSK-3α, and MISP, which favored GSK-3ß, were evaluated for reciprocal interactions. The differences in interactions between isoforms may help in understanding the distinct functions and regulation of the two isoforms as well as offer avenues for the development of isoform-specific strategies.

Towards a more general understanding of the algorithmic utility of recurrent connections.

Lateral and recurrent connections are ubiquitous in biological neural circuits. Yet while the strong computational abilities of feedforward networks have been extensively studied, our understanding of the role and advantages of recurrent computations that might explain their prevalence remains an important open challenge. Foundational studies by Minsky and Roelfsema argued that computations that require propagation of global information for local computation to take place would particularly benefit from the sequential, parallel nature of processing in recurrent networks. Such "tag propagation" algorithms perform repeated, local propagation of information and were originally introduced in the context of detecting connectedness, a task that is challenging for feedforward networks. Here, we advance the understanding of the utility of lateral and recurrent computation by first performing a large-scale empirical study of neural architectures for the computation of connectedness to explore feedforward solutions more fully and establish robustly the importance of recurrent architectures. In addition, we highlight a tradeoff between computation time and performance and construct hybrid feedforward/recurrent models that perform well even in the presence of varying computational time limitations. We then generalize tag propagation architectures to propagating multiple interacting tags and demonstrate that these are efficient computational substrates for more general computations of connectedness by introducing and solving an abstracted biologically inspired decision-making task. Our work thus clarifies and expands the set of computational tasks that can be solved efficiently by recurrent computation, yielding hypotheses for structure in population activity that may be present in such tasks.

Mapping Protein-Protein Interactions Using Data-Dependent Acquisition without Dynamic Exclusion.

Systematic analysis of affinity-purified samples by liquid chromatography coupled to mass spectrometry (LC-MS) requires high coverage, reproducibility, and sensitivity. While data-independent acquisition (DIA) approaches improve the reproducibility of protein-protein interaction detection as compared to standard data-dependent acquisition approaches, the need for library generation reduces their throughput, and analysis pipelines are still being optimized. In this study, we report the development of a simple and robust approach, termed turboDDA, to improve interactome analysis using spectral counting and data-dependent acquisition (DDA) by eliminating the dynamic exclusion (DE) step and optimizing the acquisition parameters. Using representative interaction and proximity proteomics samples, we detected increases in identified interactors of 18-71% compared to all samples analyzed by standard DDA with dynamic exclusion and for most samples analyzed by DIA with the MSPLIT-DIA spectral counting approach. In summary, turboDDA provides better sensitivity and identifies more high-confident interactors than the optimized DDA with DE and comparable or better sensitivity than DIA spectral counting approaches.

Functional characterization of a PROTAC directed against BRAF mutant V600E.

The RAF family kinases function in the RAS-ERK pathway to transmit signals from activated RAS to the downstream kinases MEK and ERK. This pathway regulates cell proliferation, differentiation and survival, enabling mutations in RAS and RAF to act as potent drivers of human cancers. Drugs targeting the prevalent oncogenic mutant BRAF(V600E) have shown great efficacy in the clinic, but long-term effectiveness is limited by resistance mechanisms that often exploit the dimerization-dependent process by which RAF kinases are activated. Here, we investigated a proteolysis-targeting chimera (PROTAC) approach to BRAF inhibition. The most effective PROTAC, termed P4B, displayed superior specificity and inhibitory properties relative to non-PROTAC controls in BRAF(V600E) cell lines. In addition, P4B displayed utility in cell lines harboring alternative BRAF mutations that impart resistance to conventional BRAF inhibitors. This work provides a proof of concept for a substitute to conventional chemical inhibition to therapeutically constrain oncogenic BRAF.

Variability in Streptavidin-Sepharose Matrix Quality Can Significantly Affect Proximity-Dependent Biotinylation (BioID) Data.

Due to their ease of use and high binding affinity, streptavidin-based purification tools have become widely used for isolating biotinylated compounds from complex mixtures. We and others routinely use streptavidin-sepharose matrices to isolate biotinylated polypeptides generated in proximity-dependent biotinylation approaches, such as BioID or APEX. However, we noted sporadic, substantial variation in the quality of BioID experiments performed in the same laboratories over time, using seemingly identical protocols. Identifying the source of this problem, here, we highlight considerable variability in streptavidin contamination derived from different production lots of streptavidin-sepharose beads from the same manufacturer and demonstrate that high levels of streptavidin peptide contamination can have detrimental effects on BioID data. We also describe two simple, rapid approaches to assess the degree of streptavidin "shedding" from individual lots of the sepharose matrix before use to avoid the use of lower quality reagent.

Neprosin, a Selective Prolyl Endoprotease for Bottom-up Proteomics and Histone Mapping.

Trypsin dominates bottom-up proteomics, but there are reasons to consider alternative enzymes. Improving sequence coverage, exposing proteomic "dark matter," and clustering post-translational modifications in different ways and with higher-order drive the pursuit of reagents complementary to trypsin. Additionally, enzymes that are easy to use and generate larger peptides that capitalize upon newer fragmentation technologies should have a place in proteomics. We expressed and characterized recombinant neprosin, a novel prolyl endoprotease of the DUF239 family, which preferentially cleaves C-terminal to proline residues under highly acidic conditions. Cleavage also occurs C-terminal to alanine with some frequency, but with an intriguingly high "skipping rate." Digestion proceeds to a stable end point, resulting in an average peptide mass of 2521 units and a higher dependence upon electron-transfer dissociation for peptide-spectrum matches. In contrast to most proline-cleaving enzymes, neprosin effectively degrades proteins of any size. For 1251 HeLa cell proteins identified in common using trypsin, Lys-C, and neprosin, almost 50% of the neprosin sequence contribution is unique. The high average peptide mass coupled with cleavage at residues not usually modified provide new opportunities for profiling clusters of post-translational modifications. We show that neprosin is a useful reagent for reading epigenetic marks on histones. It generates peptide 1-38 of histone H3 and peptide 1-32 of histone H4 in a single digest, permitting the analysis of co-occurring post-translational modifications in these important N-terminal tails.

DIA-Umpire: comprehensive computational framework for data-independent acquisition proteomics.

As a result of recent improvements in mass spectrometry (MS), there is increased interest in data-independent acquisition (DIA) strategies in which all peptides are systematically fragmented using wide mass-isolation windows ('multiplex fragmentation'). DIA-Umpire (http://diaumpire.sourceforge.net/), a comprehensive computational workflow and open-source software for DIA data, detects precursor and fragment chromatographic features and assembles them into pseudo-tandem MS spectra. These spectra can be identified with conventional database-searching and protein-inference tools, allowing sensitive, untargeted analysis of DIA data without the need for a spectral library. Quantification is done with both precursor- and fragment-ion intensities. Furthermore, DIA-Umpire enables targeted extraction of quantitative information based on peptides initially identified in only a subset of the samples, resulting in more consistent quantification across multiple samples. We demonstrated the performance of the method with control samples of varying complexity and publicly available glycoproteomics and affinity purification-MS data.

Assessment of a method to characterize antibody selectivity and specificity for use in immunoprecipitation.

Antibodies are used in multiple cell biology applications, but there are no standardized methods to assess antibody quality-an absence that risks data integrity and reproducibility. We describe a mass spectrometry-based standard operating procedure for scoring immunoprecipitation antibody quality. We quantified the abundance of all the proteins in immunoprecipitates of 1,124 new recombinant antibodies for 152 chromatin-related human proteins by comparing normalized spectral abundance factors from the target antigen with those of all other proteins. We validated the performance of the standard operating procedure in blinded studies in five independent laboratories. Antibodies for which the target antigen or a member of its known protein complex was the most abundant protein were classified as 'IP gold standard'. This method generates quantitative outputs that can be stored and archived in public databases, and it represents a step toward a platform for community benchmarking of antibody quality.

Mapping differential interactomes by affinity purification coupled with data-independent mass spectrometry acquisition.

Characterizing changes in protein-protein interactions associated with sequence variants (e.g., disease-associated mutations or splice forms) or following exposure to drugs, growth factors or hormones is critical to understanding how protein complexes are built, localized and regulated. Affinity purification (AP) coupled with mass spectrometry permits the analysis of protein interactions under near-physiological conditions, yet monitoring interaction changes requires the development of a robust and sensitive quantitative approach, especially for large-scale studies in which cost and time are major considerations. We have coupled AP to data-independent mass spectrometric acquisition (sequential window acquisition of all theoretical spectra, SWATH) and implemented an automated data extraction and statistical analysis pipeline to score modulated interactions. We used AP-SWATH to characterize changes in protein-protein interactions imparted by the HSP90 inhibitor NVP-AUY922 or melanoma-associated mutations in the human kinase CDK4. We show that AP-SWATH is a robust label-free approach to characterize such changes and propose a scalable pipeline for systems biology studies.

BDNF signaling in the VTA links the drug-dependent state to drug withdrawal aversions.

Drug administration to avoid unpleasant drug withdrawal symptoms has been hypothesized to be a crucial factor that leads to compulsive drug-taking behavior. However, the neural relationship between the aversive motivational state produced by drug withdrawal and the development of the drug-dependent state still remains elusive. It has been observed that chronic exposure to drugs of abuse increases brain-derived neurotrophic factor (BDNF) levels in ventral tegmental area (VTA) neurons. In particular, BDNF expression is dramatically increased during drug withdrawal, which would suggest a direct connection between the aversive state of withdrawal and BDNF-induced neuronal plasticity. Using lentivirus-mediated gene transfer to locally knock down the expression of the BDNF receptor tropomyosin-receptor-kinase type B in rats and mice, we observed that chronic opiate administration activates BDNF-related neuronal plasticity in the VTA that is necessary for both the establishment of an opiate-dependent state and aversive withdrawal motivation. Our findings highlight the importance of a bivalent, plastic mechanism that drives the negative reinforcement underlying addiction.

Albumin decrease is associated with spontaneous preterm delivery within 48 h in women with threatened preterm labor.

Threatened preterm labor (TPTL) accounts for ∼30% of pregnancy-related hospital admissions. Maternal peripheral leukocytes can be used to monitor a variety of physiological processes occurring in the body. Two high-throughput mass spectrometry methodologies, SWATH and iTRAQ, were used to study differentially expressed peripheral blood leukocyte lysate proteins in symptomatic women admitted for TPTL who had a preterm birth within 48 h (n = 16) and those who did not (n = 24). The SWATH spectral library consisted of 783 proteins. SWATH methodology quantified 258 proteins (using ≥2 peptides) and 5 proteins (ALBU, ANXA6, HNRPK, HSP90A, and PDIA1) were differentially expressed (p < 0.05, Mann-Whitney U). iTRAQ workflow identified 765 proteins; 354 proteins were quantified and 14 proteins (MIF, UBIQ, HXK3, ALBU, HNRPD, ST1A2, RS15A, RAP1B, CAN1, IQGA2, ST1A1, COX5A, ADDA, and UBQL1) were significantly different between the two groups of women (p < 0.05, Mann-Whitney U). Albumin was the only common differentially expressed protein in both SWATH (28% decrease) and iTRAQ studies (45% decrease). This decrease in albumin was validated using ELISA (11% decrease, p < 0.05, Mann-Whitney U) in another 23 TPTL women. This work suggests that albumin is a broad indicator of leukocyte activation with impending preterm birth and provides new future work directions to understand the pathophysiology of TPTL.

Novel NEDD1 phosphorylation sites regulate γ-tubulin binding and mitotic spindle assembly.

During cell division, microtubules organize a bipolar spindle to drive accurate chromosome segregation to daughter cells. Microtubules are nucleated by the γ-TuRC, a γ-tubulin complex that acts as a template for microtubules with 13 protofilaments. Cells lacking γ-TuRC core components do nucleate microtubules; however, these polymers fail to form bipolar spindles. NEDD1 is a γ-TuRC-interacting protein whose depletion, although not affecting γ-TuRC stability, causes spindle defects similar to the inhibition of its core subunits, including γ-tubulin. Several residues of NEDD1 are phosphorylated in mitosis. However, previously identified phosphorylation sites only partially regulate NEDD1 function, as NEDD1 depletion has a much stronger phenotype than mutation of these residues. Using mass spectrometry, we have identified multiple novel phosphorylated sites in the serine (S)557-S574 region of NEDD1, close to its γ-tubulin-binding domain. Serine to alanine mutations in S565-S574 inhibit the binding of NEDD1 to γ-tubulin and perturb NEDD1 mitotic function, yielding microtubule organization defects equivalent to those observed in NEDD1-depleted cells. Interestingly, additional mutations in the S557-T560 region restore the capacity of NEDD1 to bind γ-tubulin and promote bipolar spindle assembly. All together, our data suggest that the NEDD1/γ-tubulin interaction is finely tuned by multiple phosphorylation events in the S557-S574 region and is critical for spindle assembly. We also found that CEP192, a centrosomal protein similarly required for spindle formation, associates with NEDD1 and modulates its mitotic phosphorylation. Thus CEP192 may regulate spindle assembly by modulating NEDD1 function.

SAINT: probabilistic scoring of affinity purification-mass spectrometry data.

We present 'significance analysis of interactome' (SAINT), a computational tool that assigns confidence scores to protein-protein interaction data generated using affinity purification-mass spectrometry (AP-MS). The method uses label-free quantitative data and constructs separate distributions for true and false interactions to derive the probability of a bona fide protein-protein interaction. We show that SAINT is applicable to data of different scales and protein connectivity and allows transparent analysis of AP-MS data.

Rapid and sensitive MRM-based mass spectrometry approach for systematically exploring ganglioside-protein interactions.

Gangliosides are ubiquitous components of cell membranes. Their interactions with bacterial toxins and membrane-associated proteins (e.g. receptor tyrosine kinases) have important roles in the regulation of multiple cellular functions. Currently, an effective approach for measuring ganglioside-protein interactions especially in a large-scale fashion is largely missing. To this end, we report a facile MS-based approach to explore gangliosides extracted from cells and measure their interactions with protein of interest globally. We optimized a two-step protocol for extracting total gangliosides from cells within 2 h. Easy-to-use magnetic beads conjugated with a protein of interest were used to capture interacting gangliosides. To measure ganglioside-protein interaction on a global scale, we applied a high-sensitive LC-MS system, containing hydrophilic interaction LC separation and multiple reaction monitoring-based MS for ganglioside detection. Sensitivity for ganglioside GM1 is below 100 pg, and the whole analysis can be done in 20 min with isocratic elution. To measure ganglioside interactions with soluble vascular endothelial growth factor receptor 1 (sFlt1), we extracted and readily detected 36 species of gangliosides from perivascular retinal pigment epithelium cells across eight different classes. Twenty-three ganglioside species have significant interactions with sFlt1 as compared with IgG control based on p value cutoff <0.05. These results show that the described method provides a rapid and high-sensitive approach for systematically measuring ganglioside-protein interactions.

Identification of triazenyl indoles as inhibitors of fungal fatty acid biosynthesis with broad-spectrum activity.

Rising drug resistance among pathogenic fungi, paired with a limited antifungal arsenal, poses an increasing threat to human health. To identify antifungal compounds, we screened the RIKEN natural product depository against representative isolates of four major human fungal pathogens. This screen identified NPD6433, a triazenyl indole with broad-spectrum activity against all screening strains, as well as the filamentous mold Aspergillus fumigatus. Mechanistic studies indicated that NPD6433 targets the enoyl reductase domain of fatty acid synthase 1 (Fas1), covalently inhibiting its flavin mononucleotide-dependent NADPH-oxidation activity and arresting essential fatty acid biosynthesis. Robust Fas1 inhibition kills Candida albicans, while sublethal inhibition impairs diverse virulence traits. At well-tolerated exposures, NPD6433 extended the lifespan of nematodes infected with azole-resistant C. albicans. Overall, identification of NPD6433 provides a tool with which to explore lipid homeostasis as a therapeutic target in pathogenic fungi and reveals a mechanism by which Fas1 function can be inhibited.