A Workflow for Improved Analysis of Cross-Linking Mass Spectrometry Data Integrating Parallel Accumulation-Serial Fragmentation with MeroX and Skyline.

Cross-linking mass spectrometry (XL-MS) has evolved into a pivotal technique for probing protein interactions. This study describes the implementation of Parallel Accumulation-Serial Fragmentation (PASEF) on timsTOF instruments, enhancing the detection and analysis of protein interactions by XL-MS. Addressing the challenges in XL-MS, such as the interpretation of complex spectra, low abundant cross-linked peptides, and a data acquisition bias, our current study integrates a peptide-centric approach for the analysis of XL-MS data and presents the foundation for integrating data-independent acquisition (DIA) in XL-MS with a vendor-neutral and open-source platform. A novel workflow is described for processing data-dependent acquisition (DDA) of PASEF-derived information. For this, software by Bruker Daltonics is used, enabling the conversion of these data into a format that is compatible with MeroX and Skyline software tools. Our approach significantly improves the identification of cross-linked products from complex mixtures, allowing the XL-MS community to overcome current analytical limitations.

Delineating organizational principles of the endogenous L-A virus by cryo-EM and computational analysis of native cell extracts.

The high abundance of most viruses in infected host cells benefits their structural characterization. However, endogenous viruses are present in low copy numbers and are therefore challenging to investigate. Here, we retrieve cell extracts enriched with an endogenous virus, the yeast L-A virus. The determined cryo-EM structure discloses capsid-stabilizing cation-π stacking, widespread across viruses and within the Totiviridae, and an interplay of non-covalent interactions from ten distinct capsomere interfaces. The capsid-embedded mRNA decapping active site trench is supported by a constricting movement of two flexible opposite-facing loops. tRNA-loaded polysomes and other biomacromolecules, presumably mRNA, are found in virus proximity within the cell extract. Mature viruses participate in larger viral communities resembling their rare in-cell equivalents in terms of size, composition, and inter-virus distances. Our results collectively describe a 3D-architecture of a viral milieu, opening the door to cell-extract-based high-resolution structural virology.

RAVER1 hinders lethal EMT and modulates miR/RISC activity by the control of alternative splicing.

The RAVER1 protein serves as a co-factor in guiding the polypyrimidine tract-binding protein (PTBP)-dependent control of alternative splicing (AS). Whether RAVER1 solely acts in concert with PTBPs and how it affects cancer cell fate remained elusive. Here, we provide the first comprehensive investigation of RAVER1-controlled AS in cancer cell models. This reveals a pro-oncogenic role of RAVER1 in modulating tumor growth and epithelial-mesenchymal-transition (EMT). Splicing analyses and protein-association studies indicate that RAVER1 guides AS in association with other splicing regulators, including PTBPs and SRSFs. In cancer cells, one major function of RAVER1 is the stimulation of proliferation and restriction of apoptosis. This involves the modulation of AS events within the miR/RISC pathway. Disturbance of RAVER1 impairs miR/RISC activity resulting in severely deregulated gene expression, which promotes lethal TGFB-driven EMT. Among others, RAVER1-modulated splicing events affect the insertion of protein interaction modules in factors guiding miR/RISC-dependent gene silencing. Most prominently, in all three human TNRC6 proteins, RAVER1 controls AS of GW-enriched motifs, which are essential for AGO2-binding and the formation of active miR/RISC complexes. We propose, that RAVER1 is a key modulator of AS events in the miR/RISC pathway ensuring proper abundance and composition of miR/RISC effectors. This ensures balanced expression of TGFB signaling effectors and limits TGFB induced lethal EMT.

Structural surfaceomics reveals an AML-specific conformation of integrin ß2 as a CAR T cellular therapy target.

Safely expanding indications for cellular therapies has been challenging given a lack of highly cancer-specific surface markers. Here we explore the hypothesis that tumor cells express cancer-specific surface protein conformations that are invisible to standard target discovery pipelines evaluating gene or protein expression, and these conformations can be identified and immunotherapeutically targeted. We term this strategy integrating cross-linking mass spectrometry with glycoprotein surface capture 'structural surfaceomics'. As a proof of principle, we apply this technology to acute myeloid leukemia (AML), a hematologic malignancy with dismal outcomes and no known optimal immunotherapy target. We identify the activated conformation of integrin ß2 as a structurally defined, widely expressed AML-specific target. We develop and characterize recombinant antibodies to this protein conformation and show that chimeric antigen receptor T cells eliminate AML cells and patient-derived xenografts without notable toxicity toward normal hematopoietic cells. Our findings validate an AML conformation-specific target antigen and demonstrate a tool kit for applying these strategies more broadly.

The structural principles underlying molybdenum insertase complex assembly.

Within the cell, the trace element molybdenum (Mo) is only biologically active when complexed either within the nitrogenase-specific FeMo cofactor or within the molybdenum cofactor (Moco). Moco consists of an organic part, called molybdopterin (MPT) and an inorganic part, that is, the Mo-center. The enzyme which catalyzes the Mo-center formation is the molybdenum insertase (Mo-insertase). Mo-insertases consist of two functional domains called G- and E-domain. The G-domain catalyzes the formation of adenylated MPT (MPT-AMP), which is the substrate for the E-domain, that catalyzes the actual molybdate insertion reaction. Though the functions of E- and G-domain have been elucidated to great structural and mechanistic detail, their combined function is poorly characterized. In this work, we describe a structural model of the eukaryotic Mo-insertase Cnx1 complex that was generated based on cross-linking mass spectrometry combined with computational modeling. We revealed Cnx1 to form an asymmetric hexameric complex which allows the E- and G-domain active sites to align in a catalytic productive orientation toward each other.

Structural assessment of the full-length wild-type tumor suppressor protein p53 by mass spectrometry-guided computational modeling.

The tetrameric tumor suppressor p53 represents a great challenge for 3D-structural analysis due to its high degree of intrinsic disorder (ca. 40%). We aim to shed light on the structural and functional roles of p53's C-terminal region in full-length, wild-type human p53 tetramer and their importance for DNA binding. For this, we employed complementary techniques of structural mass spectrometry (MS) in an integrated approach with computational modeling. Our results show no major conformational differences in p53 between DNA-bound and DNA-free states, but reveal a substantial compaction of p53's C-terminal region. This supports the proposed mechanism of unspecific DNA binding to the C-terminal region of p53 prior to transcription initiation by specific DNA binding to the core domain of p53. The synergies between complementary structural MS techniques and computational modeling as pursued in our integrative approach is envisioned to serve as general strategy for studying intrinsically disordered proteins (IDPs) and intrinsically disordered region (IDRs).

Pan-tumor T-lymphocyte detection using deep neural networks: Recommendations for transfer learning in immunohistochemistry.

The success of immuno-oncology treatments promises long-term cancer remission for an increasing number of patients. The response to checkpoint inhibitor drugs has shown a correlation with the presence of immune cells in the tumor and tumor microenvironment. An in-depth understanding of the spatial localization of immune cells is therefore critical for understanding the tumor's immune landscape and predicting drug response. Computer-aided systems are well suited for efficiently quantifying immune cells in their spatial context. Conventional image analysis approaches are often based on color features and therefore require a high level of manual interaction. More robust image analysis methods based on deep learning are expected to decrease this reliance on human interaction and improve the reproducibility of immune cell scoring. However, these methods require sufficient training data and previous work has reported low robustness of these algorithms when they are tested on out-of-distribution data from different pathology labs or samples from different organs. In this work, we used a new image analysis pipeline to explicitly evaluate the robustness of marker-labeled lymphocyte quantification algorithms depending on the number of training samples before and after being transferred to a new tumor indication. For these experiments, we adapted the RetinaNet architecture for the task of T-lymphocyte detection and employed transfer learning to bridge the domain gap between tumor indications and reduce the annotation costs for unseen domains. On our test set, we achieved human-level performance for almost all tumor indications with an average precision of 0.74 in-domain and 0.72-0.74 cross-domain. From our results, we derive recommendations for model development regarding annotation extent, training sample selection, and label extraction for the development of robust algorithms for immune cell scoring. By extending the task of marker-labeled lymphocyte quantification to a multi-class detection task, the pre-requisite for subsequent analyses, e.g., distinguishing lymphocytes in the tumor stroma from tumor-infiltrating lymphocytes, is met.

RUNX1 isoform disequilibrium promotes the development of trisomy 21-associated myeloid leukemia.

Gain of chromosome 21 (Hsa21) is among the most frequent aneuploidies in leukemia. However, it remains unclear how partial or complete amplifications of Hsa21 promote leukemogenesis and why children with Down syndrome (DS) (ie, trisomy 21) are particularly at risk of leukemia development. Here, we propose that RUNX1 isoform disequilibrium with RUNX1A bias is key to DS-associated myeloid leukemia (ML-DS). Starting with Hsa21-focused CRISPR-CRISPR-associated protein 9 screens, we uncovered a strong and specific RUNX1 dependency in ML-DS cells. Expression of the RUNX1A isoform is elevated in patients with ML-DS, and mechanistic studies using murine ML-DS models and patient-derived xenografts revealed that excess RUNX1A synergizes with the pathognomonic Gata1s mutation during leukemogenesis by displacing RUNX1C from its endogenous binding sites and inducing oncogenic programs in complex with the MYC cofactor MAX. These effects were reversed by restoring the RUNX1A:RUNX1C equilibrium in patient-derived xenografts in vitro and in vivo. Moreover, pharmacological interference with MYC:MAX dimerization using MYCi361 exerted strong antileukemic effects. Thus, our study highlights the importance of alternative splicing in leukemogenesis, even on a background of aneuploidy, and paves the way for the development of specific and targeted therapies for ML-DS, as well as for other leukemias with Hsa21 aneuploidy or RUNX1 isoform disequilibrium.

Identification of HMGA2 as a predictive biomarker of response to bintrafusp alfa in a phase 1 trial in patients with advanced triple-negative breast cancer.

Background: We report the clinical activity, safety, and identification of a predictive biomarker for bintrafusp alfa, a first-in-class bifunctional fusion protein composed of the extracellular domain of TGFßRII (a TGF-ß "trap") fused to a human IgG1 mAb blocking PD-L1, in patients with advanced triple-negative breast cancer (TNBC). Methods: In this expansion cohort of a global phase 1 study, patients with pretreated, advanced TNBC received bintrafusp alfa 1200 mg every 2 weeks intravenously until disease progression, unacceptable toxicity, or withdrawal. The primary objective was confirmed best overall response by RECIST 1.1 assessed per independent review committee (IRC). Results: As of May 15, 2020, a total of 33 patients had received bintrafusp alfa, for a median of 6.0 (range, 2.0-48.1) weeks. The objective response rate was 9.1% (95% CI, 1.9%-24.3%) by IRC and investigator assessment. The median progression-free survival per IRC was 1.3 (95% CI, 1.2-1.4) months, and median overall survival was 7.7 (95% CI, 2.1-10.9) months. Twenty-five patients (75.8%) experienced treatment-related adverse events (TRAEs). Grade 3 TRAEs occurred in 5 patients (15.2%); no patients had a grade 4 TRAE. There was 1 treatment-related death (dyspnea, hemolysis, and thrombocytopenia in a patient with extensive disease at trial entry). Responses occurred independently of PD-L1 expression, and tumor RNAseq data identified HMGA2 as a potential biomarker of response. Conclusions: Bintrafusp alfa showed clinical activity and manageable safety in patients with heavily pretreated advanced TNBC. HMGA2 was identified as a potential predictive biomarker of response. ClinicalTrialsgov identifier: NCT02517398.

Does filter-aided sample preparation provide sufficient method linearity for quantitative plant shotgun proteomics?

Due to its outstanding throughput and analytical resolution, gel-free LC-based shotgun proteomics represents the gold standard of proteome analysis. Thereby, the efficiency of sample preparation dramatically affects the correctness and reliability of protein quantification. Thus, the steps of protein isolation, solubilization, and proteolysis represent the principal bottleneck of shotgun proteomics. The desired performance of the sample preparation protocols can be achieved by the application of detergents. However, these compounds ultimately compromise reverse-phase chromatographic separation and disrupt electrospray ionization. Filter-aided sample preparation (FASP) represents an elegant approach to overcome these limitations. Although this method is comprehensively validated for cell proteomics, its applicability to plants and compatibility with plant-specific protein isolation protocols remain to be confirmed. Thereby, the most important gap is the absence of the data on the linearity of underlying protein quantification methods for plant matrices. To fill this gap, we address here the potential of FASP in combination with two protein isolation protocols for quantitative analysis of pea (Pisum sativum) seed and Arabidopsis thaliana leaf proteomes by the shotgun approach. For this aim, in comprehensive spiking experiments with bovine serum albumin (BSA), we evaluated the linear dynamic range (LDR) of protein quantification in the presence of plant matrices. Furthermore, we addressed the interference of two different plant matrices in quantitative experiments, accomplished with two alternative sample preparation workflows in comparison to conventional FASP-based digestion of cell lysates, considered here as a reference. The spiking experiments revealed high sensitivities (LODs of up to 4 fmol) for spiked BSA and LDRs of at least 0.6 × 102. Thereby, phenol extraction yielded slightly better recoveries, whereas the detergent-based method showed better linearity. Thus, our results indicate the very good applicability of FASP to quantitative plant proteomics with only limited impact of the protein isolation technique on the method's overall performance.

Direct Observation of "Elongated" Conformational States in α-Synuclein upon Liquid-Liquid Phase Separation.

α-Synuclein (α-syn) is an intrinsically disordered protein (IDP) that undergoes liquid-liquid phase separation (LLPS), fibrillation, and forms insoluble intracellular Lewy bodies in neurons, which are the hallmark of Parkinson's Disease (PD). Neurotoxicity precedes the formation of aggregates and might be related to α-syn LLPS. The molecular mechanisms underlying the early stages of LLPS are still elusive. To obtain structural insights into α-syn upon LLPS, we take advantage of cross-linking/mass spectrometry (XL-MS) and introduce an innovative approach, termed COMPASS (COMPetitive PAiring StatisticS). In this work, we show that the conformational ensemble of α-syn shifts from a "hairpin-like" structure towards more "elongated" conformational states upon LLPS. We obtain insights into the critical initial stages of LLPS and establish a novel mass spectrometry-based approach that will aid to solve open questions in LLPS structural biology.

Azide- and diazirine-modified membrane lipids: Physicochemistry and applicability to study peptide/lipid interactions via cross-linking/mass spectrometry.

Although the incorporation of photo-activatable lipids into membranes potentially opens new avenues for studying interactions with peptides and proteins, the question of whether azide- or diazirine-modified lipids are suitable for such studies remains controversial. We have recently shown that diazirine-modified lipids can indeed form cross-links to membrane peptides after UV activation and that these cross-links can be precisely determined in their position by mass spectrometry (MS). However, we also observed an unexpected backfolding of the lipid's diazirine-containing stearoyl chain to the membrane interface challenging the potential application of this modified lipid for future cross-linking (XL)-MS studies of protein/lipid interactions. In this work, we compared an azide- (AzidoPC) and a diazirine-modified (DiazPC) membrane lipid regarding their self-assembly properties, their mixing behavior with saturated bilayer-forming phospholipids, and their reactivity upon UV activation using differential scanning calorimetry (DSC), dynamic light scattering (DLS), small-angle X-ray scattering (SAXS), transmission electron microscopy (TEM), and MS. Mixtures of both modified lipids with DMPC were further used for photo-chemically induced XL experiments with a transmembrane model peptide (KLAW23) to elucidate similarities and differences between the azide and the diazirine moiety. We showed that both photo-reactive lipids can be used to study lipid/peptide and lipid/protein interactions. The AzidoPC proved easier to handle, whereas the DiazPC had fewer degradation products and a higher cross-linking yield. However, the problem of backfolding occurs in both lipids; thus, it seems to be a general phenomenon.

The loss of polyphenol oxidase function is associated with hilum pigmentation and has been selected during pea domestication.

Seed coats serve as protective tissue to the enclosed embryo. As well as mechanical there are also chemical defence functions. During domestication, the property of the seed coat was altered including the removal of the seed dormancy. We used a range of genetic, transcriptomic, proteomic and metabolomic approaches to determine the function of the pea seed polyphenol oxidase (PPO) gene. Sequencing analysis revealed one nucleotide insertion or deletion in the PPO gene, with the functional PPO allele found in all wild pea samples, while most cultivated peas have one of the three nonfunctional ppo alleles. PPO functionality cosegregates with hilum pigmentation. PPO gene and protein expression, as well as enzymatic activity, was downregulated in the seed coats of cultivated peas. The functionality of the PPO gene relates to the oxidation and polymerisation of gallocatechin in the seed coat. Additionally, imaging mass spectrometry supports the hypothesis that hilum pigmentation is conditioned by the presence of both phenolic precursors and sufficient PPO activity. Taken together these results indicate that the nonfunctional polyphenol oxidase gene has been selected during pea domestication, possibly due to better seed palatability or seed coat visual appearance.

Different Oligomeric States of the Tumor Suppressor p53 Show Identical Binding Behavior towards the S100ß Homodimer.

The tumor suppressor protein p53 is a transcription factor that is referred to as the "guardian of the genome" and plays an important role in cancer development. p53 is active as a homotetramer; the S100ß homodimer binds to the intrinsically disordered C-terminus of p53 affecting its transcriptional activity. The p53/S100ß complex is regarded as highly promising therapeutic target in cancer. It has been suggested that S100ß exerts its oncogenic effects by altering the p53 oligomeric state. Our aim was to study the structures and oligomerization behavior of different p53/S100ß complexes by ESI-MS, XL-MS, and SPR. Wild-type p53 and single amino acid variants, representing different oligomeric states of p53 were individually investigated regarding their binding behavior towards S100ß. The stoichiometry of the different p53/S100ß complexes were determined by ESI-MS showing that tetrameric, dimeric, and monomeric p53 variants all bind to an S100ß dimer. In addition, XL-MS revealed the topologies of the p53/S100ß complexes to be independent of p53's oligomeric state. With SPR, the thermodynamic parameters were determined for S100ß binding to tetrameric, dimeric, or monomeric p53 variants. Our data prove that the S100ß homodimer binds to different oligomeric states of p53 with similar binding affinities. This emphasizes the need for alternative explanations to describe the molecular mechanisms underlying p53/S100ß interaction.

Reconstitution of 3' end processing of mammalian pre-mRNA reveals a central role of RBBP6.

The 3' ends of almost all eukaryotic mRNAs are generated in an essential two-step processing reaction: endonucleolytic cleavage of an extended precursor followed by the addition of a poly(A) tail. By reconstituting the reaction from overproduced and purified proteins, we provide a minimal list of 14 polypeptides that are essential and two that are stimulatory for RNA processing. In a reaction depending on the polyadenylation signal AAUAAA, the reconstituted system cleaves pre-mRNA at a single preferred site corresponding to the one used in vivo. Among the proteins, cleavage factor I stimulates cleavage but is not essential, consistent with its prominent role in alternative polyadenylation. RBBP6 is required, with structural data showing it to contact and presumably activate the endonuclease CPSF73 through its DWNN domain. The C-terminal domain of RNA polymerase II is dispensable. ATP, but not its hydrolysis, supports RNA cleavage by binding to the hClp1 subunit of cleavage factor II with submicromolar affinity.

Modulation of transcriptional mineralocorticoid receptor activity by casein kinase 1.

The mineralocorticoid receptor (MR) with its ligand aldosterone (aldo) physiologically regulates electrolyte homeostasis and blood pressure but it can also lead to pathophysiological effects in the cardiovascular system. Previous results show that posttranslational modifications (PTM) can influence MR signaling and function. Based on in silico and in vitro data, casein kinase 1 (CK1) was predicted as a candidate for MR phosphorylation. To gain a deeper mechanistic insight into MR activation, we investigated the influence of CK1 on MR function in HEK cells. Co-immunoprecipitation experiments indicated that the MR is located in a protein-protein complex with CK1α and CK1ε. Reporter gene assays with pharmacological inhibitors and MR constructs demonstrated that especially CK1ε acts as a positive modulator of GRE activity via the C-terminal MR domains CDEF. CK1 enhanced the binding affinity of aldosterone to the MR, facilitated nuclear translocation and DNA interaction of the MR, and led to expression changes of pathophysiologically relevant genes like Per-1 and Phlda1. By peptide microarray and site-directed mutagenesis experiments, we identified the highly conserved T800 as a direct CK1 phosphorylation site of the MR, which modulates the nuclear import and genomic activity of the receptor. Direct phosphorylation of the MR was unable to fully account for all of the CK1 effects on MR signaling, suggesting additional phosphorylation of MR co-regulators. By LC/MS/MS, we identified the MR-associated proteins NOLC1 and TCOF1 as candidates for such CK1-regulated co-factors. Overall, we found that CK1 acts as a co-activator of MR GRE activity through direct and indirect phosphorylation, which accelerates cytosolic-nuclear trafficking, facilitates nuclear accumulation and DNA binding of the MR, and increases the expression of pathologically relevant MR-target genes.

The megakaryocytic transcription factor ARID3A suppresses leukemia pathogenesis.

Given the plasticity of hematopoietic stem and progenitor cells, multiple routes of differentiation must be blocked in the the pathogenesis of acute myeloid leukemia, the molecular basis of which is incompletely understood. We report that posttranscriptional repression of the transcription factor ARID3A by miR-125b is a key event in the pathogenesis of acute megakaryoblastic leukemia (AMKL). AMKL is frequently associated with trisomy 21 and GATA1 mutations (GATA1s), and children with Down syndrome are at a high risk of developing the disease. The results of our study showed that chromosome 21-encoded miR-125b synergizes with Gata1s to drive leukemogenesis in this context. Leveraging forward and reverse genetics, we uncovered Arid3a as the main miR-125b target behind this synergy. We demonstrated that, during normal hematopoiesis, this transcription factor promotes megakaryocytic differentiation in concert with GATA1 and mediates TGFß-induced apoptosis and cell cycle arrest in complex with SMAD2/3. Although Gata1s mutations perturb erythroid differentiation and induce hyperproliferation of megakaryocytic progenitors, intact ARID3A expression assures their megakaryocytic differentiation and growth restriction. Upon knockdown, these tumor suppressive functions are revoked, causing a blockade of dual megakaryocytic/erythroid differentiation and subsequently of AMKL. Inversely, restoring ARID3A expression relieves the arrest of megakaryocytic differentiation in AMKL patient-derived xenografts. This work illustrates how mutations in lineage-determining transcription factors and perturbation of posttranscriptional gene regulation can interact to block multiple routes of hematopoietic differentiation and cause leukemia. In AMKL, surmounting this differentiation blockade through restoration of the tumor suppressor ARID3A represents a promising strategy for treating this lethal pediatric disease.

Native mass spectrometry identifies the HybG chaperone as carrier of the Fe(CN)2CO group during maturation of E. coli [NiFe]-hydrogenase 2.

[NiFe]-hydrogenases activate dihydrogen. Like all [NiFe]-hydrogenases, hydrogenase 2 of Escherichia coli has a bimetallic NiFe(CN)2CO cofactor in its catalytic subunit. Biosynthesis of the Fe(CN)2CO group of the [NiFe]-cofactor occurs on a distinct scaffold complex comprising the HybG and HypD accessory proteins. HybG is a member of the HypC-family of chaperones that confers specificity towards immature hydrogenase catalytic subunits during transfer of the Fe(CN)2CO group. Using native mass spectrometry of an anaerobically isolated HybG-HypD complex we show that HybG carries the Fe(CN)2CO group. Our results also reveal that only HybG, but not HypD, interacts with the apo-form of the catalytic subunit. Finally, HybG was shown to have two distinct, and apparently CO2-related, covalent modifications that depended on the presence of the N-terminal cysteine residue on the protein, possibly representing intermediates during Fe(CN)2CO group biosynthesis. Together, these findings suggest that the HybG chaperone is involved in both biosynthesis and delivery of the Fe(CN)2CO group to its target protein. HybG is thus suggested to shuttle between the assembly complex and the apo-catalytic subunit. This study provides new insights into our understanding of how organometallic cofactor components are assembled on a scaffold complex and transferred to their client proteins.

Impact of DICER1 and DROSHA on the Angiogenic Capacity of Human Endothelial Cells.

RNAi-mediated knockdown of DICER1 and DROSHA, enzymes critically involved in miRNA biogenesis, has been postulated to affect the homeostasis and the angiogenic capacity of human endothelial cells. To re-evaluate this issue, we reduced the expression of DICER1 or DROSHA by RNAi-mediated knockdown and subsequently investigated the effect of these interventions on the angiogenic capacity of human umbilical vein endothelial cells (HUVEC) in vitro (proliferation, migration, tube formation, endothelial cell spheroid sprouting) and in a HUVEC xenograft assay in immune incompetent NSGTM mice in vivo. In contrast to previous reports, neither knockdown of DICER1 nor knockdown of DROSHA profoundly affected migration or tube formation of HUVEC or the angiogenic capacity of HUVEC in vivo. Furthermore, knockdown of DICER1 and the combined knockdown of DICER1 and DROSHA tended to increase VEGF-induced BrdU incorporation and induced angiogenic sprouting from HUVEC spheroids. Consistent with these observations, global proteomic analyses showed that knockdown of DICER1 or DROSHA only moderately altered HUVEC protein expression profiles but additively reduced, for example, expression of the angiogenesis inhibitor thrombospondin-1. In conclusion, global reduction of miRNA biogenesis by knockdown of DICER1 or DROSHA does not inhibit the angiogenic capacity of HUVEC. Further studies are therefore needed to elucidate the influence of these enzymes in the context of human endothelial cell-related angiogenesis.

High-throughput characterization of photocrosslinker-bearing ion channel variants to map residues critical for function and pharmacology.

Incorporation of noncanonical amino acids (ncAAs) can endow proteins with novel functionalities, such as crosslinking or fluorescence. In ion channels, the function of these variants can be studied with great precision using standard electrophysiology, but this approach is typically labor intensive and low throughput. Here, we establish a high-throughput protocol to conduct functional and pharmacological investigations of ncAA-containing human acid-sensing ion channel 1a (hASIC1a) variants in transiently transfected mammalian cells. We introduce 3 different photocrosslinking ncAAs into 103 positions and assess the function of the resulting 309 variants with automated patch clamp (APC). We demonstrate that the approach is efficient and versatile, as it is amenable to assessing even complex pharmacological modulation by peptides. The data show that the acidic pocket is a major determinant for current decay, and live-cell crosslinking provides insight into the hASIC1a-psalmotoxin 1 (PcTx1) interaction. Further, we provide evidence that the protocol can be applied to other ion channels, such as P2X2 and GluA2 receptors. We therefore anticipate the approach to enable future APC-based studies of ncAA-containing ion channels in mammalian cells.