https://www.nature.com/articles/s41586-022-04727-9

Proper ectodermal patterning during human development requires previously identified transcription factors such as GATA3 and p63, as well as positional signalling from regional mesoderm1-6. However, the mechanism by which ectoderm and mesoderm factors act to stably pattern gene expression and lineage commitment remains unclear. Here we identify the protein Gibbin, encoded by the Xia-Gibbs AT-hook DNA-binding-motif-containing 1 (AHDC1) disease gene7-9, as a key regulator of early epithelial morphogenesis. We find that enhancer- or promoter-bound Gibbin interacts with dozens of sequence-specific zinc-finger transcription factors and methyl-CpG-binding proteins to regulate the expression of mesoderm genes. The loss of Gibbin causes an increase in DNA methylation at GATA3-dependent mesodermal genes, resulting in a loss of signalling between developing dermal and epidermal cell types. Notably, Gibbin-mutant human embryonic stem-cell-derived skin organoids lack dermal maturation, resulting in p63-expressing basal cells that possess defective keratinocyte stratification. In vivo chimeric CRISPR mouse mutants reveal a spectrum of Gibbin-dependent developmental patterning defects affecting craniofacial structure, abdominal wall closure and epidermal stratification that mirror patient phenotypes. Our results indicate that the patterning phenotypes seen in Xia-Gibbs and related syndromes derive from abnormal mesoderm maturation as a result of gene-specific DNA methylation decisions.

(Protein Metrics). Proteolysis was assumed to be tryptic allowing for N-ragged cleavage with up to two missed cleavage sites. Precursor and fragment mass accuracies Save

https://www.sciencedirect.com/science/article/abs/pii/S1097276522003215?via%3Dihub

The p53 transcription factor drives anti-proliferative gene expression programs in response to diverse stressors, including DNA damage and oncogenic signaling. Here, we seek to uncover new mechanisms through which p53 regulates gene expression using tandem affinity purification/mass spectrometry to identify p53-interacting proteins. This approach identified METTL3, an m6A RNA-methyltransferase complex (MTC) constituent, as a p53 interactor. We find that METTL3 promotes p53 protein stabilization and target gene expression in response to DNA damage and oncogenic signals, by both catalytic activity-dependent and independent mechanisms. METTL3 also enhances p53 tumor suppressor activity in in vivo mouse cancer models and human cancer cells. Notably, METTL3 only promotes tumor suppression in the context of intact p53. Analysis of human cancer genome data further supports the notion that the MTC reinforces p53 function in human cancer. Together, these studies reveal a fundamental role for METTL3 in amplifying p53 signaling in response to cellular stress.

Enrichr Chen et al. (2013), Kuleshov et al. (2016) https://maayanlab.cloud/Enrichr/ Metascape Zhou et al. (2019) http://metascape.org/gp/index.html#/main/step1 Byonic Protein Metrics https://proteinmetrics.com/resources/byonic-advanced-peptide-and-protein-identification-software/

https://tuprints.ulb.tu-darmstadt.de/21046/

Iron is an essential transition metal required in cell culture medium (CCM) due to its vital role in many cellular processes such as energy metabolism, deoxyribonucleic acid (DNA) biosynthesis or antioxidant functions. However, due to its redox capabilities, iron can also catalyze Fenton reactions favoring the formation of reactive oxygen species (ROS) that may cause severe cellular damages. This study sought to investigate the impact of iron in CCM on Chinese hamster ovary (CHO) cell line performance, on critical quality attributes (CQAs) of different recombinant proteins, on messenger ribonucleic acid (mRNA) expression levels of genes involved in iron homeostasis, and on intracellular iron or labile iron pool (LIP) levels, whereby for the last two readouts a method development was performed prior to analyzing the project relevant samples. Besides the successful establishment of a ferrozine-based assay for detecting total intracellular iron amount and the promising results obtained upon testing the fluorescent probe RhoNox-1 for detecting changes in LIP concentrations, none of the other two (fluorescent) probes tested during method development, namely calcein and TRX-PURO, were able to determine LIP amounts within CHO cells. Since those probes rely on a rather high LIP concentration present within cells, a low LIP present within CHO cells either due to a limited, saturated or low iron uptake, or due to an immediate distribution or usage of iron within the cells once taken up was thus suggested. Data also revealed that iron raw material impurities are strongly impacting cell performance and CQAs. Whereas manganese was identified as the main impurity improving cell performance and altering protein glycosylation level within Cellvento 4CHO and 4Feed fed-batch platform with manganese presenting additionally an opposite effect on cell culture compared to iron, copper impurity contributed to an overall increased cell performance of the tested CHOZN cell line in EX-CELL Advanced CHO Fed-Batch-Medium platform. Usage of low impurity iron raw materials is therefore crucial to decouple the effects of iron and its trace element impurities by controlling and adjusting each element concentration independently and thereby guarantee the run of consistent and stable cell culture processes. Among the different iron sources tested within CCM during a fed-batch experiment, non-chelated iron sources caused a faster decrease in measured iron concentration within the supernatant and led to a higher detected iron amount present within the cell pellets taken during the course of the fed-batch process compared to the tested chelated iron sources, whereas no cell growth was obtained upon ferric chloride (FeCl3) usage. At a first glance, data suggest an increased uptake efficiency for CHO cells upon usage of non-chelated iron sources, however, differences might have rather come from a faster iron precipitate formation within CCM upon usage of non-chelated iron sources, additionally, since mRNA expression levels of genes involved in iron uptake did not indicate for a difference between chelated and non-chelated iron sources. The removal of possible iron precipitates prior to intracellular iron measurement as well as the investigation of the fate of iron in CCM seems thus to be crucial to understand iron-related uptake mechanisms.

Byonic™ 3.1 Protein Metrics

https://pubs.acs.org/doi/full/10.1021/acs.bioconjchem.1c00572

N-linked glycosylation is one of the most common and complex posttranslational modifications that govern the biological functions and physicochemical properties of therapeutic antibodies. We evaluated thermal and metabolic stabilities of antibody-drug conjugates (ADCs) with payloads attached to the C’E loop in the immunoglobulin G (IgG) Fc CH2 domain, comparing the glycosylated and aglycosylated Fc ADC variants. Our study revealed that introduction of small-molecule drugs into an aglycosylated antibody can compensate for thermal destabilization originating from structural distortions caused by elimination of N-linked glycans. Depending on the conjugation site, glycans had both positive and negative effects on plasma stability of ADCs. The findings highlight the importance of consideration for selection of conjugation site to achieve desirable physicochemical properties and plasma stability.

stion mixture is further captured by addition of a second set of beads coated with anti-human Fc capture reagent.24 The MS data were analyzed following deconvolution using Intact Mass software (v3.6, Protein Metrics, Inc.) The relative peak intensities of parent (ADC fragment with acetyl payload) and catabolite (ADC fragment with deacetyl payload) were used to determine the relative percent deacet;- (24) The MS data were analyzed following deconvolution using Intact Mass software (v3.6, Protein Metrics, Inc.) The relative peak intensities of parent (ADC fragment with Save)