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Vitamin B12 (cobalamin) is required for most human gut microbes, many of which are dependent on scavenging to obtain this vitamin. Since bacterial densities in the gut are extremely high, competition for this keystone micronutrient is severe. Contrasting with Enterobacteria, members of the dominant genus Bacteroides often encode several BtuB vitamin B12 outer membrane transporters together with a conserved array of surface-exposed B12-binding lipoproteins. Here we show that the BtuB transporters from Bacteroides thetaiotaomicron form stable, pedal bin-like complexes with surface-exposed BtuG lipoprotein lids, which bind B12 with high affinities. Closing of the BtuG lid following B12 capture causes destabilisation of the bound B12 by a conserved BtuB extracellular loop, causing translocation of the vitamin to BtuB and subsequent transport. We propose that TonB-dependent, lipoprotein-assisted small molecule uptake is a general feature of Bacteroides spp. that is important for the success of this genus in colonising the human gut. Uptake of vitamin B12 is important for colonisation of the gut by Bacteroides species. Here the authors characterise B12 uptake in Bacteroides thetaiotaomicron, mediated by outer membrane protein complexes consisting of surface-exposed BtuG lipoproteins and BtuB TonB-dependent transporters. [ABSTRACT FROM AUTHOR]

Introduction and aims CYLD cutaneous syndrome (CCS) is an inherited hair follicle tumour syndrome where biallelic loss of function of the deubiquitinase enzyme CYLD gives rise to cylindroma skin tumours. The role of CYLD in skin biology is not well characterized but it is known to regulate several cell signalling pathways through deubiquitination of effector proteins. We aimed to determine how loss of CYLD function can lead to CCS skin tumour formation by studying the ubiquitome of CCS tumours and healthy skin. Methods Healthy skin (n = 5) and CCS tumour tissue (n = 5) were collected from flash frozen samples. Lysed homogenates were trypsin-digested and the resulting peptides underwent diglycine immunopurification for subsequent liquid chromatography–tandem mass spectrometry analysis on a Bruker Tims-TOF HT (Billerica, MA, USA) mass spectrometer. We utilized a data-independent acquisition method adapted for diglycine-containing peptides to generate mass spectrometry data. Peptide identification was performed by searching mass spectrometry data against an in silico neural network derived spectral library with diglycine modification. Results We identified 1273 ubiquitination sites across 649 proteins in CCS tumour and control samples. After performing t -tests (P < 0.05), we found 305 ubiquitination sites that were either unique to their respective condition or were differentially regulated. Analysis of ubiquitinated peptide sequences uncovered cell surface receptors with increased ubiquitination in CCS tumour samples including the insulin receptor, which had ubiquitination modifications at its kinase domain, suggesting increased insulin signalling in CCS tumours. Gene set enrichment analysis of CCS tumour ubiquitinated substrates revealed increased ubiquitination of secretory granules and extracellular exosomes, suggesting that loss of CYLD may disrupt regulation of protein secretion processes. Conclusions Our findings describe the ubiquitome of CCS keratinocytes, providing insights into how loss of CYLD function can affect biological processes in skin. [ABSTRACT FROM AUTHOR]

The engulfment of "self" and "non-self" particles by immune and non-immune cells is crucial for maintaining homeostasis and combatting infection. Engulfed particles are contained within vesicles termed phagosomes that undergo dynamic fusion and fission events, which ultimately results in the formation of phagolysosomes that degrade the internalized cargo. This process is highly conserved and plays an important role in maintaining homeostasis, and disruptions in this are implicated in numerous inflammatory disorders. Given its broad role in innate immunity, it is important to understand how different stimuli or changes within the cell can shape the phagosome architecture. In this chapter, we describe a robust protocol for the isolation of polystyrene bead-induced phagosomes using sucrose density gradient centrifugation. This process results in a highly pure sample that can be used in downstream applications, namely, Western blotting., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)