Degradation of Airway Secretory Cell Mucin Granules Is Dependent on Lysosome Activity

Inflammatory airway diseases (e.g. COPD and asthma) are associated with mucous cell metaplasia and mucin hypersecretion, resulting in symptoms such as shortness of breath and cough. However, how secretory cells remove excess mucin granules is poorly understood. Previous research suggests that intracellular degradation pathways, such as autophagy, are involved in the degradation of mucin granules during resolution of mucous cell metaplasia. We thus hypothesized that the elimination of excess mucin granules is dependent on lysosome-mediated degradation in airway secretory cells. Calu-3 cells, an airway epithelial cell line containing abundant mucin granules, were treated with inhibitors of lysosome acidification (Bafilomycin A1) and lysosome enzyme activity (Pepstatin E64d). We found statistically significant increases in the levels of secretory mucin, MUC5AC, by mucin blot, suggesting that the lysosome mediates the elimination of mucin granules. In addition, by immunoblot we observed an increase in the autophagosome markers, LC3-II and SQSTM1, with lysosome inhibition using Bafilomycin A1, indicating an accumulation of autophagosomes and a role for autophagy in the degradation of mucin granules. However, after transfecting Calu-3 cells with a ubiquitin-hemagglutinin tag plasmid to examine the role of the proteasome in the degradation of mucin granules, we observed that our transfection efficiency was low, making it difficult to detect the hemagglutinin epitope by immunoblots. Nevertheless, we found that MUC5AC levels preliminarily increase with the inhibition of the proteasome using MG-132, suggesting a potential role for the proteasome in the degradation of mucin granules. Thus, we can conclude that inhibition of the lysosome increases MUC5AC levels, demonstrating that the lysosome mediates the degradation of mucin granules in airway secretory cells. In addition, while we were not able to conclude that the proteasome is involved in the degradation of mucin granules with certainty, our preliminary data suggests that it is possible that the ubiquitin-proteasome system is involved in the degradation of mucin due to the observed increase in MUC5AC levels with MG-132.https://digitalcommons.unmc.edu/surp2022/1039/thumbnail.jp

A multi-scale analysis of bull sperm methylome revealed both species peculiarities and conserved tissue-specific

peer-reviewedBackground: Spermatozoa have a remarkable epigenome in line with their degree of specialization, their unique nature and different requirements for successful fertilization. Accordingly, perturbations in the establishment of DNA methylation patterns during male germ cell differentiation have been associated with infertility in several species.Background: Spermatozoa have a remarkable epigenResults: The quantification of DNA methylation at CCGG sites using luminometric methylation assay (LUMA) highlighted the undermethylation of bull sperm compared to the sperm of rams, stallions, mice, goats and men. Total blood cells displayed a similarly high level of methylation in bulls and rams, suggesting that undermethylation of the bovine genome was specific to sperm. Annotation of CCGG sites in different species revealed no striking bias in the distribution of genome features targeted by LUMA that could explain undermethylation of bull sperm. To map DNA methylation at a genome-wide scale, bull sperm was compared with bovine liver, fibroblasts and monocytes using reduced representation bisulfite sequencing (RRBS) and immunoprecipitation of methylated DNA followed by microarray hybridization (MeDIP-chip). These two methods exhibited differences in terms of genome coverage, and consistently, two independent sets of sequences differentially methylated in sperm and somatic cells were identified for RRBS and MeDIP-chip. Remarkably, in the two sets most of the differentially methylated sequences were hypomethylated in sperm. In agreement with previous studies in other species, the sequences that were specifically hypomethylated in bull sperm targeted processes relevant to the germline differentiation program (piRNA metabolism, meiosis, spermatogenesis) and sperm functions (cell adhesion, fertilization), as well as satellites and rDNA repeats. Conclusions: These results highlight the undermethylation of bull spermatozoa when compared with both bovine somatic cells and the sperm of other mammals, and raise questions regarding the dynamics of DNA methylation in bovine male germline. Whether sperm undermethylation has potential interactions with structural variation in the cattle genome may deserve further attention. While bull semen is widely used in artificial insemination, the literature describing DNA methylation in bull spermatozoa is still scarce. The purpose of this study was therefore to characterize the bull sperm methylome relative to both bovine somatic cells and the sperm of other mammals through a multiscale analysis

Bovine sperm plasma membrane proteomics through biotinylation and subcellular enrichment

A significant proportion of mammalian fertilization is mediated through the proteomic composition of the sperm surface. These protein constituents can present as biomarkers to control and regulate breeding of agricultural animals. Previous studies have addressed the bovine sperm cell apical plasma membrane (PM) proteome with nitrogen cavitation enrichment. Alternative workflows would enable to expand the compositional data more globally around the entire sperm's surface. We used a cell surface biotin‐labeling in combination with differential centrifugation to enrich sperm surface proteins. Using nano‐LC MS/MS, 338 proteins were confidently identified in the PM‐enriched proteome. Functional categories of sperm–egg interaction, protein turnover, metabolism as well as molecular transport, spermatogenesis, and signal transduction were represented by proteins with high quantitative signal in our study. A highly significant degree of enrichment was found for transmembrane and PM‐targeted proteins. Among them, we also report proteins previously not described on bovine sperm (CPQ, CD58, CKLF, CPVL, GLB1L3, and LPCAT2B) of which CPQ and CPVL cell surface localization was further validated. A descriptive overview of the bovine sperm PM integral and peripheral proteins is provided to complement future studies on animal reproduction and its relation to sperm cell surface. All MS data have been deposited in the ProteomeXchange with identifier PXD001096 (http://proteomecentral.proteomexchange.org/dataset/PXD001096)