Human fetal brain self-organizes into long-term expanding organoids.

Human brain development involves an orchestrated, massive neural progenitor expansion while a multi-cellular tissue architecture is established. Continuously expanding organoids can be grown directly from multiple somatic tissues, yet to date, brain organoids can solely be established from pluripotent stem cells. Here, we show that healthy human fetal brain in vitro self-organizes into organoids (FeBOs), phenocopying aspects of in vivo cellular heterogeneity and complex organization. FeBOs can be expanded over long time periods. FeBO growth requires maintenance of tissue integrity, which ensures production of a tissue-like extracellular matrix (ECM) niche, ultimately endowing FeBO expansion. FeBO lines derived from different areas of the central nervous system (CNS), including dorsal and ventral forebrain, preserve their regional identity and allow to probe aspects of positional identity. Using CRISPR-Cas9, we showcase the generation of syngeneic mutant FeBO lines for the study of brain cancer. Taken together, FeBOs constitute a complementary CNS organoid platform.

Genotype-dependent N-glycosylation and newly exposed O-glycosylation affect plasmin-induced cleavage of histidine-rich glycoprotein (HRG).

Histidine-rich glycoprotein (HRG) is an abundant plasma protein harboring at least three N-glycosylation sites. HRG integrates many biological processes, such as coagulation, antiangiogenic activity, and pathogen clearance. Importantly, HRG is known to exhibit five genetic variants with minor allele frequencies of more than 10%. Among them, Pro204Ser can induce a fourth N-glycosylation site (Asn202). Considerable efforts have been made to reveal the biological function of HRG, whereas data on HRG glycosylation are scarcer. To close this knowledge gap, we used C18-based LC-MS/MS to study the glycosylation characteristics of six HRG samples from different sources. We used endogenous HRG purified from human plasma and compared its glycosylation to that of the recombinant HRG produced in Chinese hamster ovary cells or human embryonic kidney 293 cells, targeting distinct genotypic isoforms. In endogenous plasma HRG, every N-glycosylation site was occupied predominantly with a sialylated diantennary complex-type glycan. In contrast, in the recombinant HRGs, all glycans showed different antennarities, sialylation, and core fucosylation, as well as the presence of oligomannose glycans, LacdiNAcs, and antennary fucosylation. Furthermore, we observed two previously unreported O-glycosylation sites in HRG on residues Thr273 and Thr274. These sites together showed more than 90% glycan occupancy in all HRG samples studied. To investigate the potential relevance of HRG glycosylation, we assessed the plasmin-induced cleavage of HRG under various conditions. These analyses revealed that the sialylation of the N- and O-glycans as well as the genotype-dependent N-glycosylation significantly influenced the kinetics and specificity of plasmin-induced cleavage of HRG.

The pleiotropic phenotype of FlbA of Aspergillus niger is explained in part by the activity of seven of its downstream-regulated transcription factors.

Inactivation of flbA in Aspergillus niger results in thinner cell walls, increased cell lysis, abolished sporulation, and an increased secretome complexity. A total of 36 transcription factor (TF) genes are differentially expressed in ΔflbA. Here, seven of these genes (abaA, aslA, aslB, azf1, htfA, nosA, and srbA) were inactivated. Inactivation of each of these genes affected sporulation and, with the exception of abaA, cell wall integrity and protein secretion. The impact on secretion was strongest in the case of ΔaslA and ΔaslB that showed increased pepsin, cellulase, and amylase activity. Biomass was reduced of agar cultures of ΔabaA, ΔaslA, ΔnosA, and ΔsrbA, while biomass was higher in liquid shaken cultures of ΔaslA and ΔaslB. The ΔaslA and ΔhtfA strains showed increased resistance to H2O2, while ΔaslB was more sensitive to this reactive oxygen species. Together, inactivation of the seven TF genes impacted biomass formation, sporulation, protein secretion, and stress resistance, and thereby these genes explain at least part of the pleiotropic phenotype of ΔflbA of A. niger.

Glycoproteomics-Compatible MS/MS-Based Quantification of Glycopeptide Isomers.

Glycosylation is an essential protein modification occurring on the majority of extracellular human proteins, with mass spectrometry (MS) being an indispensable tool for its analysis, that not only determines glycan compositions, but also the position of the glycan at specific sites via glycoproteomics. However, glycans are complex branching structures with monosaccharides interconnected in a variety of biologically relevant linkages, isomeric properties that are invisible when the readout is mass alone. Here, we developed an LC-MS/MS-based workflow for determining glycopeptide isomer ratios. Making use of isomerically defined glyco(peptide) standards, we observed marked differences in fragmentation behavior between isomer pairs when subjected to collision energy gradients, specifically in terms of the galactosylation/sialylation branching and linkage. These behaviors were developed into component variables that allowed for relative quantification of isomerism within mixtures. Importantly, at least for small peptides, the isomer quantification appeared to be largely independent from the peptide portion of the conjugate, allowing a broad application of the method.

Novel Components of the Stress Assembly Sec Body Identified by Proximity Labeling.

Sec bodies are membraneless stress-induced assemblies that form by the coalescence of endoplasmic reticulum exit sites (ERES). Through APEX2 tagging of Sec24AB, we biotinylated and identified the full complement of Sec body proteins. In the presence of biotin-phenol and H2O2 (APEX on), APEX2 facilitates the transfer of a biotin moiety to nearby interactors of chimeric Sec24AB. Using this unbiased approach comparing APEX on and off (-H2O2) conditions, we identified 52 proteins specifically enriched in Sec bodies. These include a large proportion of ER and Golgi proteins, packaged without defined stoichiometry, which we could selectively verify by imaging. Interestingly, Sec body components are neither transcriptionally nor translationally regulated under the conditions that induce Sec body formation, suggesting that incorporation of these proteins into granules may be driven instead by the aggregation of nucleating proteins with a high content of intrinsically disordered regions. This reinforces the notion that Sec bodies may act as storage for ERES, ER and Golgi components during stress.

GAGE mediates radio resistance in cervical cancers via the regulation of chromatin accessibility.

Radiotherapy (RT) resistance is a major cause of treatment failure in cancers that use definitive RT as their primary treatment modality. This study identifies the cancer/testis (CT) antigen G antigen (GAGE) as a mediator of radio resistance in cervical cancers. Elevated GAGE expression positively associates with de novo RT resistance in clinical samples. GAGE, specifically the GAGE12 protein variant, confers RT resistance through synemin-dependent chromatin localization, promoting the association of histone deacetylase 1/2 (HDAC1/2) to its inhibitor actin. This cumulates to elevated histone 3 lysine 56 acetylation (H3K56Ac) levels, increased chromatin accessibility, and improved DNA repair efficiency. Molecular or pharmacological disruption of the GAGE-associated complex restores radiosensitivity. Molecularly, this study demonstrates the role of GAGE in the regulation of chromatin dynamics. Clinically, this study puts forward the utility of GAGE as a pre-screening biomarker to identify poor responders at initial diagnosis and the therapeutic potential of agents that target GAGE and its associated complex in combination with radiotherapy to improve outcomes.

Cysteine Deprivation Targets Ovarian Clear Cell Carcinoma Via Oxidative Stress and Iron-Sulfur Cluster Biogenesis Deficit.

Aims: Current treatment options for ovarian clear cell carcinoma (OCCC) are limited to combination of platinum-based and other cytotoxic agents to which patients respond poorly due to intrinsic chemoresistance. There is therefore an urgent need to develop alternative therapeutic strategies for OCCC. Results: Cysteine deprivation suppresses OCCC growth in vitro and in vivo with no apparent toxicity. Modes of cell death induced by cysteine deprivation in OCCC are determined by their innate metabolic profiles. Cysteine-deprived glycolytic OCCC is abolished primarily by oxidative stress-dependent necrosis and ferroptosis, which can otherwise be prevented by pretreatment with antioxidative agents. Meanwhile, OCCC that relies on mitochondria respiration for its bioenergetics is suppressed through apoptosis, which can otherwise be averted by pretreatment with cysteine precursor alone, but not with antioxidative agents. Cysteine deprivation induces apoptosis in respiring OCCC by limiting iron-sulfur (Fe-S) cluster synthesis in the mitochondria, without which electron transport chain may be disrupted. Respiring OCCC responds to Fe-S cluster deficit by increasing iron influx into the mitochondria, which leads to iron overload, mitochondria damage, and eventual cell death. Innovation/Conclusion: This study demonstrates the importance of cysteine availability in OCCC that is for its antioxidative property and its less appreciated role in mitochondria respiration. Regardless of OCCC metabolic profiles, cysteine deprivation abolishes both glycolytic and respiring OCCC growth in vitro and in vivo. Conclusion: This study highlights the therapeutic potential of cysteine deprivation for OCCC.

A Proteomics Approach to Identify New Putative Cardiac Intercalated Disk Proteins.

AIMS: Synchronous beating of the heart is dependent on the efficient functioning of the cardiac intercalated disk (ID). The ID is composed of a complex protein network enabling electrical continuity and chemical communication between individual cardiomyocytes. Recently, several different studies have shed light on increasingly prevalent cardiac diseases involving the ID. Insufficient knowledge of its composition makes it difficult to study these disease mechanisms in more detail and therefore here we aim expand the ID proteome. Here, using a combination of general membrane enrichment, in-depth quantitative proteomics and an intracellular location driven bioinformatics approach, we aim to discover new putative ID proteins in rat ventricular tissue. METHODS AND RESULTS: General membrane isolation, enriched amongst others also with ID proteins as based on presence of the established markers connexin-43 and n-cadherin, was performed using centrifugation. By mass spectrometry, we quantitatively evaluated the level of 3455 proteins in the enriched membrane fraction (EMF) and its counterpart, the soluble cytoplasmic fraction. These data were stringently filtered to generate a final set of 97 enriched, putative ID proteins. These included Cx43 and n-cadherin, but also many interesting novel candidates. We selected 4 candidates (Flotillin-2 (FLOT2), Nexilin (NEXN), Popeye-domain-containg-protein 2 (POPDC2) and thioredoxin-related-transmembrane-protein 2 (TMX2)) and confirmed their co-localization with n-cadherin in the ID of human and rat heart cryo-sections, and isolated dog cardiomyocytes. CONCLUSION: The presented proteomics dataset of putative new ID proteins is a valuable resource for future research into this important molecular intersection of the heart.

Alterations in the interactome of serine/threonine protein phosphatase type-1 in atrial fibrillation patients.

BACKGROUND: Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia, yet current pharmacological treatments are limited. Serine/threonine protein phosphatase type-1 (PP1), a major phosphatase in the heart, consists of a catalytic subunit (PP1c) and a large set of regulatory (R)-subunits that confer localization and substrate specificity to the holoenzyme. Previous studies suggest that PP1 is dysregulated in AF, but the mechanisms are unknown. OBJECTIVES: The purpose of this study was to test the hypothesis that PP1 is dysregulated in paroxysmal atrial fibrillation (PAF) at the level of its R-subunits. METHODS: Cardiac lysates were coimmunoprecipitated with anti-PP1c antibody followed by mass spectrometry-based, quantitative profiling of associated R-subunits. Subsequently, label-free quantification (LFQ) was used to evaluate altered R-subunit-PP1c interactions in PAF patients. R-subunits with altered binding to PP1c in PAF were further studied using bioinformatics, Western blotting (WB), immunocytochemistry, and coimmunoprecipitation. RESULTS: A total of 135 and 78 putative PP1c interactors were captured from mouse and human cardiac lysates, respectively, including many previously unreported interactors with conserved PP1c docking motifs. Increases in binding were found between PP1c and PPP1R7, cold-shock domain protein A (CSDA), and phosphodiesterase type-5A (PDE5A) in PAF patients, with CSDA and PDE5A being novel interactors validated by bioinformatics, immunocytochemistry, and coimmunoprecipitation. WB confirmed that these increases in binding cannot be ascribed to their changes in global protein expression alone. CONCLUSIONS: Subcellular heterogeneity in PP1 activity and downstream protein phosphorylation in AF may be attributed to alterations in PP1c-R-subunit interactions, which impair PP1 targeting to proteins involved in electrical and Ca(2+) remodeling. This represents a novel concept in AF pathogenesis and may provide more specific drug targets for treating AF.

CD98 marks a subpopulation of head and neck squamous cell carcinoma cells with stem cell properties.

Patients with advanced head and neck squamous cell carcinomas (HNSCCs) are often treated with concomitant chemotherapy and radiotherapy, but only 50% is cured. A possible explanation for treatment failure is therapy resistance of the cancer stem cells (CSCs). The application of compounds specifically targeting these CSCs, in addition to routinely used therapeutics, would likely improve clinical outcome. We demonstrate that the previously described monoclonal antibody K984 recognizes the CD98 cell surface protein, which is specifically expressed by cells forming the squamous basal cell layer, the region where the squamous stem cells reside. Moreover, CD98 is highly resistant to the proteolytic enzymes required for CSC enrichment procedures. We show that CD98(high) cells, in contrast to CD98(low) cells, are able to generate tumors in immunodeficient mice, indicating that CD98(high) cells have stem cell characteristics. Furthermore, the CD98(high) subpopulation expresses high levels of cell cycle control and DNA repair genes, while the CD98(low) fraction shows expression patterns that represent the more differentiated cells forming the bulk of the tumor. CD98 is a promising CSC enrichment marker in HNSCC. Our data support the CSC concept in head and neck cancer and the potential relevance of these cells for treatment outcome.

Protein signatures associated with tumor cell dissemination in head and neck cancer.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common type of cancer worldwide. Strong prognostic indicators that predict development of distant metastases are the presence and number of lymph node metastases in the neck, and extranodal spread. Recently, it was shown in several studies that also the presence of disseminated tumor cells (DTC) in the bone marrow predicts development of distant metastases. We have investigated whether protein signatures could be detected in primary HNSCC that distinguish tumors that disseminate into the bone marrow from those that do not. Therefore, DTC-positive and -negative primary HNSCC tumors were analyzed by 2D-DIGE. A signature consisting of 51 differential protein spots was identified upon stratification for bone marrow status, which allowed a correct classification of DTC-positive and DTC-negative HNSCC tumors in 95% of cases, using hierarchical clustering. The most prominent feature within this signature was the down-regulation of CK19 in DTC-positive tumors. Our data show that tumor cell dissemination to the bone marrow, the onset of hematogenic metastasis, can be deduced from the protein profile in the primary tumor. The highly significant down-regulation of CK19 supports a model of epithelial-mesenchymal transition for tumors that show a high proclivity for hematogenic dissemination.

Photocrosslinking and click chemistry enable the specific detection of proteins interacting with phospholipids at the membrane interface.

New lipid analogs mimicking the abundant membrane phospholipid phosphatidylcholine were developed to photocrosslink proteins interacting with phospholipid headgroups at the membrane interface. In addition to either a phenylazide or benzophenone photoactivatable moiety attached to the headgroup, the lipid analogs contained azides attached as baits to the acyl chains. After photocrosslinking in situ in the biomembrane, these baits were used for the attachment of a fluorescent tetramethylrhodamine-alkyne conjugate or a biotin-alkyne conjugate using click chemistry, allowing for the selective detection and purification of crosslink products, respectively. Proteins crosslinked to the lipid analogs in inner mitochondrial membranes from Saccharomyces cerevisiae were detected and subsequently identified by mass spectrometry. Established interaction partners of phosphatidylcholine were found, as well as known integral and peripheral inner membrane proteins, and proteins that were not previously considered mitochondrial inner membrane proteins.

NK cell protease granzyme M targets alpha-tubulin and disorganizes the microtubule network.

Serine protease granzyme M (GrM) is highly expressed in the cytolytic granules of NK cells, which eliminate virus-infected cells and tumor cells. The molecular mechanisms by which GrM induces cell death, however, remain poorly understood. In this study we used a proteomic approach to scan the native proteome of human tumor cells for intracellular substrates of GrM. Among other findings, this approach revealed several components of the cytoskeleton. GrM directly and efficiently cleaved the actin-plasma membrane linker ezrin and the microtubule component alpha-tubulin by using purified proteins, tumor cell lysates, and tumor cells undergoing cell death induced by perforin and GrM. These cleavage events occurred independently of caspases or other cysteine proteases. Kinetically, alpha-tubulin was more efficiently cleaved by GrM as compared with ezrin. Direct alpha-tubulin proteolysis by GrM is complex and occurs at multiple cleavage sites, one of them being Leu at position 269. GrM disturbed tubulin polymerization dynamics in vitro and induced microtubule network disorganization in tumor cells in vivo. We conclude that GrM targets major components of the cytoskeleton that likely contribute to NK cell-induced cell death.

Probing the membrane interface-interacting proteome using photoactivatable lipid cross-linkers.

To analyze proteins interacting at the membrane interface, a phospholipid analogue was used with a photoactivatable headgroup (ASA-DLPE, N-(4-azidosalicylamidyl)-1,2-dilauroyl-sn-glycero-3-phosphoethanolamine) for selective cross-linking. The peripheral membrane protein cytochrome c from the inner mitochondrial membrane was rendered carbonate wash-resistant by cross-linking to ASA-DLPE in a model membrane system, validating our approach. Cross-link products of cytochrome c and its precursor apocytochrome c were demonstrated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and were specifically detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), taking advantage of the intrinsic UV absorbance of the cross-linker. Application of the method to inner mitochondrial membranes from Saccharomyces cerevisae revealed cross-link products of both exogenously added apocytochrome c and endogenous proteins with molecular weights around 34 and 72 kDa. Liquid chromatograpy (LC)-MS/MS was performed to identify these proteins, resulting in a list of candidate proteins potentially cross-linked at the membrane interface. The approach described here provides methodology for capturing phospholipid-protein interactions in their native environment of the biomembrane using modern proteomics techniques.

Structural characterization and immunogenicity in wild-type and immune tolerant mice of degraded recombinant human interferon alpha2b.

PURPOSE: This study was conducted to study the influence of protein structure on the immunogenicity in wild-type and immune tolerant mice of well-characterized degradation products of recombinant human interferon alpha2b (rhIFNalpha2b). METHODS: RhIFNalpha2b was degraded by metal-catalyzed oxidation (M), cross-linking with glutaraldehyde (G), oxidation with hydrogen peroxide (H), and incubation in a boiling water bath (B). The products were characterized with UV absorption, circular dichroism and fluorescence spectroscopy, gel permeation chromatography, reverse-phase high-pressure liquid chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis, Western blotting, and mass spectrometry. The immunogenicity of the products was evaluated in wild-type mice and in transgenic mice immune tolerant for hIFNalpha2. Serum antibodies were detected by enzyme-linked immunosorbent assay or surface plasmon resonance. RESULTS: M-rhIFNalpha2b contained covalently aggregated rhIFNalpha2b with three methionines partly oxidized to methionine sulfoxides. G-rhIFNalpha2b contained covalent aggregates and did not show changes in secondary structure. H-rhIFNalpha2b was only chemically changed with four partly oxidized methionines. B-rhIFNalpha2b was largely unfolded and heavily aggregated. Nontreated (N) rhIFNalpha2b was immunogenic in the wild-type mice but not in the transgenic mice, showing that the latter were immune tolerant for rhIFNalpha2b. The anti-rhIFNalpha2b antibody levels in the wild-type mice depended on the degradation product: M-rhIFNalpha2b > H-rhIFNalpha2b approximately N-rhIFNalpha2b >> B-rhIFNalpha2b; G-rhIFNalpha2b did not induce anti-rhIFNalpha2b antibodies. In the transgenic mice, only M-rhIFNalpha2b could break the immune tolerance. CONCLUSIONS: RhIFNalpha2b immunogenicity is related to its structural integrity. Moreover, the immunogenicity of aggregated rhIFNalpha2b depends on the structure and orientation of the constituent protein molecules and/or on the aggregate size.