Sequencing of cancer genomes has identified recurrent somatic mutations in histones, termed oncohistones, which are frequently poorly understood. Previously we showed that fission yeast expressing ...only the H3.3G34R mutant identified in aggressive pediatric glioma had reduced H3K36 trimethylation and acetylation, increased genomic instability and replicative stress, and defective homology-dependent DNA damage repair. Here we show that surprisingly distinct phenotypes result from G34V (also in glioma) and G34W (giant cell tumors of bone) mutations, differentially affecting H3K36 modifications, subtelomeric silencing, genomic stability; sensitivity to irradiation, alkylating agents, and hydroxyurea; and influencing DNA repair. In cancer, only 1 of 30 alleles encoding H3 is mutated. Whilst co-expression of wild-type H3 rescues most G34 mutant phenotypes, G34R causes dominant hydroxyurea sensitivity, homologous recombination defects, and dominant subtelomeric silencing. Together, these studies demonstrate the complexity associated with different substitutions at even a single residue in H3 and highlight the utility of genetically tractable systems for their analysis.
Lectin microarray (LMA) is a high-sensitive glycan analysis technology used to obtain global glycomic profiles of both N- and O-glycans attached not only to purified glycoproteins but also to crude ...glycoprotein samples. Through additional use of laser microdissection (LMD) for tissue collection, we developed an LMA-based glycomic profiling technique for a specific type of cells in a tiny area of formalin-fixed paraffin-embedded (FFPE) tissue sections. This LMD-LMA method makes it possible to obtain reproducible tissue glycomic profiles that can be compared with each other, using a unified protocol for all procedures, including FFPE tissue preparation, tissue staining, protein extraction and labeling, and LMA analysis. Here, we describe the standardized LMD-LMA procedure for a "tissue glycome mapping" approach, which facilitates an in-depth understanding of region- and tissue-specific protein glycosylation. We also describe potential applications of the spatial tissue glycomic profiles, including histochemical analysis for evaluating distribution of lectin ligands and a fluorescence LMD-LMA method for cell type-selective glycomic profiling using a cell type-specific probe, composed of a lectin and an antibody. The protocols presented here will accelerate the effective utilization of FFPE tissue specimens by providing tissue glycome maps for the discovery of the biological roles and disease-related alterations of protein glycosylation.
Abstract
Mesothelioma is a highly aggressive tumour associated with asbestos exposure and is histologically classified into three types: epithelioid-type, sarcomatoid-type and biphasic-type. The ...prognosis of mesothelioma patients is poor and there is no effective molecular-targeting therapy as yet. ERC/mesothelin is a glycoprotein that is highly expressed on several types of cancers including epithelioid mesothelioma, but also expressed on normal mesothelial cells. This is a predicted reason why there is no clinically approved therapeutic antibody targeting ERC/mesothelin. In the present study, we focussed on the differential glycosylation between ERC/mesothelin present on epithelioid mesothelioma and that on normal mesothelial cells and aimed to reveal a distinct feature of epithelioid mesothelioma cells. Lectin microarray analysis of ERC/mesothelin using cells and patient specimens showed significantly stronger binding of PHA-E4 lectin, which recognizes complex-type N-glycans having a so-called bisecting-GlcNAc structure, to ERC/mesothelin from epithelioid mesothelioma cells than that from normal mesothelial cells. Further, liquid chromatography/mass spectrometry analysis on ERC/mesothelin from epithelioid mesothelioma cells confirmed the presence of a bisecting-GlcNAc attached to Asn388 of ERC/mesothelin. These results suggest that this glycoproteome could serve as a potential target for the generation of a highly selective and safe therapeutic antibody for epithelioid mesothelioma.
Graphical Abstract
The significance of glycomic profiling has been highlighted by recent findings that structural changes of glycans are observed in many diseases, including cancer. Therefore, glycomic profiling of the ...whole body (glycome mapping) under different physiopathological states may contribute to the discovery of reliable biomarkers with disease-specific alterations. To achieve this, standardization of high-throughput and in-depth analysis of tissue glycome mapping is needed. However, this is a great challenge due to the lack of analytical methodology for glycans on small amounts of endogenous glycoproteins. Here, we established a standardized method of lectin-assisted tissue glycome mapping. Formalin-fixed, paraffin-embedded tissue sections were prepared from brain, liver, kidney, spleen, and testis of two C57BL/6J mice. In total, 190 size-adjusted fragments with different morphology were serially collected from each tissue by laser microdissection and subjected to lectin microarray analysis. The results and subsequent histochemical analysis with selected lectins were highly consistent with previous reports of mass spectrometry-based N- and/or O-glycome analyses and histochemistry. This is the first report to look at both N- and O-glycome profiles of various regions within tissue sections of five different organs. This simple and reproducible mapping approach is also applicable to various disease model mice to facilitate disease-related biomarker discovery.
Molecular and cellular characteristics of the relapse-prone subset within triple-negative breast cancer (TNBC) remain unclear. Aberrant glycosylation is involved in the malignant behavior of cancer ...cells. In the present study, we aimed to reveal glycan profiles unique to relapsed TNBC patients.
Thirty TNBC patients who did not undergo neoadjuvant chemotherapy but postoperative standard adjuvant therapy from 2009 through 2016 at Juntendo Hospital were investigated. TNBC cells were resected from primary breast cancer sections of formalin-fixed surgical specimens using laser-assisted microdissection. The binding intensities of the extracted glycoproteins to 45 lectins were quantified using lectin microarray and compared between relapsed and non-relapsed patients. Immunohistochemical staining with TJA-II lectin in specimen sections was performed.
Five patients relapsed during the follow-up (range 37-123 months). Lectin microarray analysis revealed that 7 out of 45 lectins showed significant differences in binding intensity between the relapsed and the non-relapsed group. TJA-II, ACA, WFA, and BPL showed stronger binding in the relapsed group. PNGase F treatment of TNBC cell lysates suggested that TJA-II and ACA bind O-glycans. TJA-II staining of tissue sections revealed strong binding to cell surface membranes and to the cytoplasm of TNBC cells, but not to other types of cells. Significantly more TNBC cells were stained in tissue sections from relapsed than non-relapsed patients.
TNBC cells from relapsed patients showed a unique lectin reactivity, with higher levels of TJA-II (also WFA and BPL) binding than in non-relapsed patients. The results are potentially useful to develop new prognostic and therapeutic tools.
The nuclear pore complex (NPC) is a large protein assembly that perforates the nuclear envelope and provides a sole gateway for traffic between the cytoplasm and the nucleus. The NPC controls the ...nucleocytoplasmic transport by selectively allowing cargoes such as proteins and mRNA to pass through its central channel, thereby playing a vital role in protecting the nuclear component and regulating gene expression and protein synthesis. The selective transport through the NPC originates from its exquisite molecular structure featuring a large scaffold and the intrinsically disordered central channel domain, but the exact mechanism underlying the selective transport remains elusive and is the subject of various, often conflicting, hypotheses. Moreover, recent studies have suggested a new role for the NPC as a mechanosensor, where the NPC changes its channel diameter depending on the nuclear envelope tension, altering the molecular transportability through this nanopore. In this mini-review, we summarize the current understandings of the selective nature of the NPC and discuss its emerging role in cellular mechanotransduction.
Cells generate mechanical forces primarily from interactions between F-actin, cross-linking proteins, myosin motors, and other actin-binding proteins in the cytoskeleton. To understand how molecular ...interactions between the cytoskeletal elements generate forces, a number of in vitro experiments have been performed but are limited in their ability to accurately reproduce the diversity of motor mobility. In myosin motility assays, myosin heads are fixed on a surface and glide F-actin. By contrast, in reconstituted gels, the motion of both myosin and F-actin is unrestricted. Because only these two extreme conditions have been used, the importance of mobility of motors for network behaviors has remained unclear. In this study, to illuminate the impacts of motor mobility on the contractile behaviors of the actin cytoskeleton, we employed an agent-based computational model based on Brownian dynamics. We find that if motors can bind to only one F-actin like myosin I, networks are most contractile at intermediate mobility. In this case, less motor mobility helps motors stably pull F-actins to generate tensile forces, whereas higher motor mobility allows F-actins to aggregate into larger clustering structures. The optimal intermediate motor mobility depends on the stall force and affinity of motors that are regulated by mechanochemical rates. In addition, we find that the role of motor mobility can vary drastically if motors can bind to a pair of F-actins. A network can exhibit large contraction with high motor mobility because motors bound to antiparallel pairs of F-actins can exert similar forces regardless of their mobility. Results from this study imply that the mobility of molecular motors may critically regulate contractile behaviors of actin networks in cells.
Cohesins containing a meiosis-specific α-kleisin subunit, RAD21L or REC8, play roles in diverse aspects of meiotic chromosome dynamics including formation of axial elements (AEs), assembly of the ...synaptonemal complex (SC), recombination of homologous chromosomes (homologs), and cohesion of sister chromatids. However, the exact functions of individual α-kleisins remain to be elucidated. Here, we examined the localization of RAD21L and REC8 within the SC by super-resolution microscopy, 3D-SIM. We found that both RAD21L and REC8 were localized at the connection sites between lateral elements (LEs) and transverse filaments (TFs) of pachynema with RAD21L locating interior to REC8 sites. RAD21L and REC8 were not symmetrical in terms of synaptic homologs, suggesting that the arrangement of different cohesins is not strictly fixed along all chromosome axes. Intriguingly, some RAD21L signals, but not REC8 signals, were observed between unsynapsed regions of AEs of zygonema as if they formed a bridge between homologs. Furthermore, the signals of recombination intermediates overlapped with those of RAD21L to a greater degree than with those of REC8. These results highlight the different properties of two meiotic α-kleisins, and strongly support the previous proposition that RAD21L is an atypical cohesin that establishes the association between homologs rather than sister chromatids.
Glycoform of mucin 1 (MUC1) in cancerous cells changes markedly with cell differentiation, and thus, qualitative detection and verification of the MUC1 glycosylation changes have potential diagnostic ...value. We have developed an ultrasensitive method to detect the changes in cholangiocarcinoma (CC), which produces MUC1, and applied it in the diagnostics development. The focused glycan analysis using 43-lectin-immobilized microarray could obtain the glycan profiles of sialylated MUC1 in 5 μL of sera. The high-throughput analysis detected disease-specific alterations of glycosylation, and the statistical analysis confirmed that use of Wisteria floribunda agglutinin (WFA) alone produced a diagnostic score sufficient for discriminating 33 CC cases from 40 hepatolithiasis patients and 48 normal controls (p < 0.0001). The CC-related glycosylation change was verified by the lectin–antibody sandwich ELISA with WFA in two cohorts: (1) 78 Opisthorchis viverrini infected patients without CC and 78 with CC, (2) 33 CC patients and 40 hepatolithiasis patients (the same cohort used for the above lectin microarray). The WFA positivity distinguished patients with CC (opisthorchiasis: p < 0.0001, odds ratio = 1.047; hepatolithiasis: p = 0.0002, odds ratio = 1.018). Sensitive detection of qualitative alterations of sialylated MUC1 glycosylation is indispensable for the development of our glycodiagnostic test for CC.
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