Effects of an acute exercise bout in hypoxia on extracellular vesicle release in healthy and prediabetic subjects

Purpose: To investigate exosome-like vesicle (ELV) plasma concentrations and markers of multivesicular body (MVB) biogenesis in skeletal muscle in response to acute exercise. Methods: Seventeen healthy (BMI: 23.5±0.5kg·m-2) and fifteen prediabetic (BMI: 27.3±1.2kg·m-2) men were randomly assigned to two groups performing an acute cycling bout in normoxia or hypoxia (FiO2 14.0%). Venous blood samples were taken before (T0), during (T30) and after (T60) exercise and biopsies from m. vastus lateralis were collected before and after exercise. Plasma ELVs were isolated by size exclusion chromatography, counted by nanoparticle tracking analysis (NTA), and characterized according to international standards, followed by expression analyses of canonical ELV markers in skeletal muscle. Results: In the healthy normoxic group, the total number of particles in the plasma increased during exercise from T0 to T30 (+313%) followed by a decrease from T30 to T60 (-53%). In the same group, an increase in TSG101, CD81 and HSP60 protein expression was measured after exercise in plasma ELVs; however, in the prediabetic group, the total number of particles in the plasma was not affected by exercise. The mRNA content of TSG101, ALIX and CD9 were upregulated in skeletal muscle after exercise in normoxia; whereas, CD9 and CD81 were downregulated in hypoxia. Conclusions: ELV plasma abundance increased in response to acute aerobic exercise in healthy subjects in normoxia, but not in prediabetic subjects, nor in hypoxia. Skeletal muscle analyses suggested that this tissue did not likely play a major role of the exercise-induced increase in circulating ELVs.

Hepatitis B Immunoglobulin Inhibits The Secretion of HBV Via Antigen-Antibody Precipitation in The Multivesicular Body

Although the main action of human hepatitis B immunoglobulin (HBIG) of neutralizing the hepatitis B virus surface antigen (HBsAg) in the serum is known, HBIG is known to be localized in the cell. However, the effect of intracellularly located HBIG is not well elucidated due to the low purity of conventional plasma-derived HBIG (cHBIG). We attempted to clarify the mechanism of action of internalized HBIG using recombinant HBIG (lenvervimab). We used HBsAg cell lines, non-HBsAg cell lines and human HBsAg-producing hepatocytes. Autophagosome lysis pathway related proteins and Rab5, calnexin, giantin, and Rab7 were used to localize HBsAg and anti-HBs-IgG in the cytoplasm with Western blot and confocal microscopy.Intracellular anti-HBs-IgG (lenvervimab and cHBIG) transported by Fc receptor-mediated endocytosis increased the autophagosomes, but there was no change in autolysis. HBsAg and anti-HBs-IgG precipitated in the cytoplasm and co-localized in the multivesicular body. HBsAg secretion in the culture medium was decreased after lenvervimab. Simultaneously, the amount of cellular HBsAg increased in the cell lines but decreased in the human hepatocytes. Furthermore, intracellular lenvervimab was not easily washed out only in the HBsAg cell lines.Lenvervimab decreases the secretion of HBsAg, and HBsAg-antibody precipitation in the multivesicular body might play an important role.

Phosphate Restriction Promotes Longevity via Activation of Autophagy and the Multivesicular Body Pathway

Nutrient limitation results in an activation of autophagy in organisms ranging from yeast, nematodes and flies to mammals. Several evolutionary conserved nutrient-sensing kinases are critical for efficient adaptation of yeast cells to glucose, nitrogen or phosphate depletion, subsequent cell-cycle exit and the regulation of autophagy. Here, we demonstrate that phosphate restriction results in a prominent extension of yeast lifespan that requires the coordinated activity of autophagy and the multivesicular body pathway, enabling efficient turnover of cytoplasmic and plasma membrane cargo. While the multivesicular body pathway was essential during the early days of aging, autophagy contributed to long-term survival at later days. The cyclin-dependent kinase Pho85 was critical for phosphate restriction-induced autophagy and full lifespan extension. In contrast, when cell-cycle exit was triggered by exhaustion of glucose instead of phosphate, Pho85 and its cyclin, Pho80, functioned as negative regulators of autophagy and lifespan. The storage of phosphate in form of polyphosphate was completely dispensable to in sustaining viability under phosphate restriction. Collectively, our results identify the multifunctional, nutrient-sensing kinase Pho85 as critical modulator of longevity that differentially coordinates the autophagic response to distinct kinds of starvation.

His Domain Protein Tyrosine Phosphatase and Rabaptin-5 couple endo-lysosomal sorting of EGFR with endosomal maturation

His Domain Protein Tyrosine Phosphatase (HD-PTP) collaborates with Endosomal Sorting Complexes Required for Transport (ESCRTs) to sort endosomal cargo into intralumenal vesicles, forming the multivesicular body. Completion of multivesicular body sorting is accompanied by maturation of the endosome into a late endosome, an event that requires inactivation of the early endosomal GTPase, Rab5. Here we show that HD-PTP links ESCRT function with endosomal maturation. HD-PTP depletion prevents multivesicular body sorting, whilst also blocking cargo from exiting Rab5-rich endosomes. HD-PTP depleted cells contain hyperphosphorylated Rabaptin-5, a cofactor for the Rab5 guanine nucleotide exchange factor, Rabex-5, though HD-PTP is unlikely to directly dephosphorylate Rabaptin-5. In addition, HD-PTP depleted cells exhibit Rabaptin-5 dependent hyperactivation of Rab5. HD-PTP binds directly to Rabaptin-5, between its Rabex-5 and Rab5 binding domains. This binding reaction involves the ESCRT-0/ESCRT-III binding site in HD-PTP and is competed by an ESCRT-III peptide. Jointly, these findings indicate that HD-PTP may alternately scaffold ESCRTs and modulate Rabex-5/Rabaptin-5 activity, thereby helping to coordinate the completion of MVB sorting with endosomal maturation.

The S100A10–AnxA2 complex is associated with the exocytosis of hepatitis B virus in intrauterine infection

Mother-to-child transmission (MTCT) is the major cause of chronic infection of hepatitis B virus (HBV) in patients. However, whether and how HBV crosses the placenta to cause infection in utero remains unclear. In this study, we investigate the mechanism as to how the HBV virions pass through layers of the trophoblast. Our data demonstrate the exocytosis of virions from the trophoblast after exposure to HBV where the endocytosed HBV virions co-localized with an S100A10/AnxA2 complex and LC3, an autophagosome membrane marker. Knockdown of either AnxA2 or S100A10 in trophoblast cells led to a reduction of the amount of exo-virus in Transwell assay. Immunohistochemistry also showed a high expression of AnxA2 and S100A10 in the placental tissue samples of HBV-infected mothers with congenital HBV-positive infants (HBV+/+). We conclude that in HBV intrauterine infection and mother-to-child transmission, a proportion of HBV hijacks autophagic protein secretion pathway and translocate across the trophoblast via S100A10/AnxA2 complex and multivesicular body (MVB)-mediated exocytosis. Our study provides a potential target for the interference of the mechanisms of HBV intrauterine infection and mother-to-child transmission.

Extracellular Vesicle Proteomes Shed Light on the Evolutionary, Interactive, and Functional Divergence of Their Biogenesis Mechanisms

Extracellular vesicles (EVs) are membranous structures containing bioactive molecules, secreted by most cells into the extracellular environment. EVs are classified by their biogenesis mechanisms into two major subtypes: ectosomes (enriched in large EVs; lEVs), budding directly from the plasma membrane, which is common in both prokaryotes and eukaryotes, and exosomes (enriched in small EVs; sEVs) generated through the multivesicular bodies via the endomembrane system, which is unique to eukaryotes. Even though recent proteomic analyses have identified key proteins associated with EV subtypes, there has been no systematic analysis, thus far, to support the general validity and utility of current EV subtype separation methods, still largely dependent on physical properties, such as vesicular size and sedimentation. Here, we classified human EV proteomic datasets into two main categories based on distinct centrifugation protocols commonly used for isolating sEV or lEV fractions. We found characteristic, evolutionarily conserved profiles of sEV and lEV proteins linked to their respective biogenetic origins. This may suggest that the evolutionary trajectory of vesicular proteins may result in a membership bias toward specific EV subtypes. Protein–protein interaction (PPI) network analysis showed that vesicular proteins formed distinct clusters with proteins in the same EV fraction, providing evidence for the existence of EV subtype-specific protein recruiters. Moreover, we identified functional modules enriched in each fraction, including multivesicular body sorting for sEV, and mitochondria cellular respiration for lEV proteins. Our analysis successfully captured novel features of EVs embedded in heterogeneous proteomics studies and suggests specific protein markers and signatures to be used as quality controllers in the isolation procedure for subtype-enriched EV fractions.

Unexpected organellar locations of ESCRT machinery in Giardia intestinalis and complex evolutionary dynamics spanning the transition to parasitism in the lineage Fornicata

Background Comparing a parasitic lineage to its free-living relatives is a powerful way to understand how that evolutionary transition to parasitism occurred. Giardia intestinalis (Fornicata) is a leading cause of gastrointestinal disease world-wide and is famous for its unusual complement of cellular compartments, such as having peripheral vacuoles instead of typical endosomal compartments. Endocytosis plays an important role in Giardia’s pathogenesis. Endosomal sorting complexes required for transport (ESCRT) are membrane-deforming proteins associated with the late endosome/multivesicular body (MVB). MVBs are ill-defined in G. intestinalis, and roles for identified ESCRT-related proteins are not fully understood in the context of its unique endocytic system. Furthermore, components thought to be required for full ESCRT functionality have not yet been documented in this species. Results We used genomic and transcriptomic data from several Fornicata species to clarify the evolutionary genome streamlining observed in Giardia, as well as to detect any divergent orthologs of the Fornicata ESCRT subunits. We observed differences in the ESCRT machinery complement between Giardia strains. Microscopy-based investigations of key components of ESCRT machinery such as GiVPS36 and GiVPS25 link them to peripheral vacuoles, highlighting these organelles as simplified MVB equivalents. Unexpectedly, we show ESCRT components associated with the endoplasmic reticulum and, for the first time, mitosomes. Finally, we identified the rare ESCRT component CHMP7 in several fornicate representatives, including Giardia and show that contrary to current understanding, CHMP7 evolved from a gene fusion of VPS25 and SNF7 domains, prior to the last eukaryotic common ancestor, over 1.5 billion years ago. Conclusions Our findings show that ESCRT machinery in G. intestinalis is far more varied and complete than previously thought, associates to multiple cellular locations, and presents changes in ESCRT complement which pre-date adoption of a parasitic lifestyle.

A single genetic locus controls both expression of DPEP1/CHMP1A and kidney disease development via ferroptosis

Genome-wide association studies (GWAS) have identified loci for kidney disease, but the causal variants, genes, and pathways remain unknown. Here we identify two kidney disease genes Dipeptidase 1 (DPEP1) and Charged Multivesicular Body Protein 1 A (CHMP1A) via the triangulation of kidney function GWAS, human kidney expression, and methylation quantitative trait loci. Using single-cell chromatin accessibility and genome editing, we fine map the region that controls the expression of both genes. Mouse genetic models demonstrate the causal roles of both genes in kidney disease. Cellular studies indicate that both Dpep1 and Chmp1a are important regulators of a single pathway, ferroptosis and lead to kidney disease development via altering cellular iron trafficking.