Histone methyltransferase Dot1L recruits O-GlcNAc transferase to target chromatin sites to regulate histone O-GlcNAcylation.

O-GlcNAc transferase (OGT) is the distinctive enzyme responsible for catalyzing O-GlcNAc addition to the serine or threonine residues of thousands of cytoplasmic and nuclear proteins involved in such basic cellular processes as DNA damage repair, RNA splicing, and transcription preinitiation and initiation complex assembly. However, the molecular mechanism by which OGT regulates gene transcription remains elusive. Using proximity labeling-based mass spectrometry, here, we searched for functional partners of OGT and identified interacting protein Dot1L, a conserved and unique histone methyltransferase known to mediate histone H3 Lys79 methylation, which is required for gene transcription, DNA damage repair, cell proliferation, and embryo development. Although this specific interaction with OGT does not regulate the enzymatic activity of Dot1L, we show that it does facilitate OGT-dependent histone O-GlcNAcylation. Moreover, we demonstrate that OGT associates with Dot1L at transcription start sites and that depleting Dot1L decreases OGT associated with chromatin globally. Notably, we also show that downregulation of Dot1L reduces the levels of histone H2B S112 O-GlcNAcylation and histone H2B K120 ubiquitination in vivo, which are associated with gene transcription regulation. Taken together, these results reveal that O-GlcNAcylation of chromatin is dependent on Dot1L.

OGT Binding Peptide-Tagged Strategy Increases Protein O-GlcNAcylation Level in E. coli.

O-GlcNAcylation is a single glycosylation of GlcNAc mediated by OGT, which regulates the function of substrate proteins and is closely related to many diseases. However, a large number of O-GlcNAc-modified target proteins are costly, inefficient, and complicated to prepare. In this study, an OGT binding peptide (OBP)-tagged strategy for improving the proportion of O-GlcNAc modification was established successfully in E. coli. OBP (P1, P2, or P3) was fused with target protein Tau as tagged Tau. Tau or tagged Tau was co-constructed with OGT into a vector expressed in E. coli. Compared with Tau, the O-GlcNAc level of P1Tau and TauP1 increased 4~6-fold. Moreover, the P1Tau and TauP1 increased the O-GlcNAc-modified homogeneity. The high O-GlcNAcylation on P1Tau resulted in a significantly slower aggregation rate than Tau in vitro. This strategy was also used successfully to increase the O-GlcNAc level of c-Myc and H2B. These results indicated that the OBP-tagged strategy was a successful approach to improve the O-GlcNAcylation of a target protein for further functional research.

Identification of a type II LacNAc specific binding lectin CMRBL from Cordyceps militaris.

The Cordyceps militaris gene CCM_03832 encodes a ricin-B like lectin. The gene was cloned and expressed in Escherichia coli, and its protein product, named CMRBL (C. militaris ricin-B like lectin), was purified by galactose affinity chromatography. Of nine different sources of erythrocytes, CMRBL showed only specific hemagglutinating activity against rat and rabbit erythrocytes with titers of 22 and 28, respectively. Glycan array analyses by the Consortium for Functional Glycomics showed that CMRBL possesses very high specific binding activity of glycans terminated with type II LacNAc (non-reducing Galß1-4GlcNAc). Compared with other well-known Gal-terminated binding lectins such as Erythrina cristagalli agglutinin, Ricinus communis agglutinin, and Jacalin, CMRBL showed better binding specificity to type II LacNAc compared the other lectins. CMRBL showed lowest binding activity to ZR-75-30 and MDA-MB-468 cell lines among five tested cell lines (H22, THP-1, MDA-MB-231, ZR-75-30, and MDA-MB-468 cells). Transfection of type II LacNAc main galactosyltransferase B4GALT3 to ZR-75-30 significantly improved CMRBL binding activity compared with control. CMRBL was also applied for testing the type II LacNAc modification of Etanercept successfully. Our data suggest that CMRBL would be a useful tool to recognize type II LacNAc, especially distinguish type II from other galactose-terminated glycans in glycan biology research.

Effects of the glycosylation of the receptor binding domain (RBD dimer)-based Covid-19 vaccine (ZF2001) on its humoral immunogenicity and immunoreactivity.

The SARS-CoV-2 spike protein receptor-binding domain (RBD), which is a key target for the development of SARS-CoV-2 neutralizing antibodies and vaccines, mediates the binding of the host receptor angiotensin-converting enzyme 2 (ACE2). However, the high heterogeneity of RBD glycoforms may lead to an incomplete neutralization effect and impact the immunogenicity of RBD-based vaccines (Ye et al., 2021). Here, our data suggested that the glycosylation significantly affected the humoral immunogenicity and immunoreactivity of the RBD-dimer-based Covid-19 vaccine (ZF2001) (Yang et al., 2021). Several deglycosylated types of ZF2001 (with sialic acid removed (ZF2001-ΔSA), sialic acid & O-glycans removed (ZF2001-ΔSA&O), N-glycans removed (ZF2001-ΔN), N- & O-glycans removed (ZF2001-ΔN&O)) were obtained by treatment with glycosidases. The binding affinity between deglycosylated types of ZF2001 and ACE2 was slightly weakened and that between deglycosylated types of ZF2001 and several monoclonal antibodies (mAbs) were also changed compared with ZF2001. The results of pseudovirus neutralization assay and binding affinity assay of all ZF2001 types revealed that the antigens with complex glycosylation had better humoral immunogenicity and immunoreactivity. Molecular dynamics simulation indicated that the more complex glycosylation of RBD corresponded to more hydrogen bonds formed between helper T-cell epitopes of RBD and major histocompatibility complex II (MHC-II). In summary, these results demonstrated that the glycosylation of RBD affects antigen presentation, humoral immunogenicity and immunoreactivity, which may be an important consideration for vaccine design and production technology.

Galectin-7 promotes cisplatin efficacy by facilitating apoptosis and G3BP1 degradation in cervical cancer.

The emergence of chemoresistance in cervical cancer is extremely challenging in chemotherapy. Oxidative stress has emerged as the regulatory factor in drug resistance, but the detailed mechanism is still unknown. Stress granules, are membrane-less ribonucleoprotein-based condensates, could enhance chemoresistance by sequestering proapoptotic proteins inhibition of cell death upon exposure to drug-induced oxidative stress. Galectin-7, a member of galectin family, exerts varied roles in tumor repression or progression in different cancers. However, its role in cervical cancer has not been sufficiently studied. Here, we found that galectin-7 promotes cisplatin (CDDP) induced apoptosis and associates with stress granule-nucleating protein G3BP1 degradation. With the treatment of cisplatin, galectin-7 could enhance apoptosis by upregulating cleaved-PARP1 and the generation of reactive oxygen species (ROS), promoting mitochondrial fission, and reducing mitochondrial membrane potential (MMP). Furthermore, galectin-7 also reduces resistance by facilitating cisplatin-induced stress granules clearance through galectin-7/RACK1/G3BP1 axis. All these data suggested that galectin-7 promotes cisplatin sensitivity, and it would be potential target for potentiating efficacy in cervical cancer chemotherapy.

Conjugated/nonconjugated alternating copolymers for enhanced NIR-II fluorescence imaging and NIR-II photothermal-ferrotherapy.

Conjugated polymers hold great promise for NIR-II fluorescence imaging (FI)-guided NIR-II photothermal therapy (PTT) due to the advantages of easy modification of chemical structures and adjustable NIR absorption. However, to make use of these advantages, it is of paramount importance to formulate conjugated polymers with excellent solubility in organic solution, great NIR-II photothermal conversion efficiency, and high NIR-II fluorescence quantum yield. Herein, a new class of conjugated/nonconjugated alternating copolymers (CNACPs) is reported by introducing nonconjugated linkers into a conjugated backbone to modulate the extinction coefficient at 1064 nm and NIR-II fluorescence quantum yield. The NIR-II absorption, NIR-II emission, and NIR-II photothermal properties of the new CNACPs were studied. Interestingly, it is observed that longer nonconjugated linkers in CNACPs result in higher NIR-II fluorescence intensity with sufficient NIR-II absorption and NIR-II photothermal ability. With these newly developed CNACPs (BBT-C6), phototheranostic nanoparticles (BBTD6/Fe@PMA) are prepared through facile nanoprecipitation using PMA-AD-PEG as an iron ion chelator for NIR-II FI-guided NIR-II PTT/ferrotherapy synergistic therapy. In vitro and in vivo, BBTD6/Fe@PMA effectively inhibited 4T1 cells and tumor progression under 1064 nm laser irradiation. Consequently, this work provides new CNACPs by incorporating nonconjugated linkers into a conjugated backbone to design more effective NIR-II fluorescence imaging and NIR-II photothermal therapy agents.

A robust spike sorting method based on the joint optimization of linear discrimination analysis and density peaks.

Spike sorting is a fundamental step in extracting single-unit activity from neural ensemble recordings, which play an important role in basic neuroscience and neurotechnologies. A few algorithms have been applied in spike sorting. However, when noise level or waveform similarity becomes relatively high, their robustness still faces a big challenge. In this study, we propose a spike sorting method combining Linear Discriminant Analysis (LDA) and Density Peaks (DP) for feature extraction and clustering. Relying on the joint optimization of LDA and DP: DP provides more accurate classification labels for LDA, LDA extracts more discriminative features to cluster for DP, and the algorithm achieves high performance after iteration. We first compared the proposed LDA-DP algorithm with several algorithms on one publicly available simulated dataset and one real rodent neural dataset with different noise levels. We further demonstrated the performance of the LDA-DP method on a real neural dataset from non-human primates with more complex distribution characteristics. The results show that our LDA-DP algorithm extracts a more discriminative feature subspace and achieves better cluster quality than previously established methods in both simulated and real data. Especially in the neural recordings with high noise levels or waveform similarity, the LDA-DP still yields a robust performance with automatic detection of the number of clusters. The proposed LDA-DP algorithm achieved high sorting accuracy and robustness to noise, which offers a promising tool for spike sorting and facilitates the following analysis of neural population activity.