Milk exosomes: beyond dietary microRNAs

Extracellular vesicles deliver a variety of cargos to recipient cells, including the delivery of cargos in dietary vesicles from bovine milk to non-bovine species. The rate of discovery in this important line of research is slowed by a controversy whether the delivery and bioactivity of a single class of vesicle cargos, microRNAs, are real or not. This opinion paper argues that the evidence in support of the bioavailability of microRNAs encapsulated in dietary exosomes outweighs the evidence produced by scholars doubting that phenomenon is real. Importantly, this paper posits that the time is ripe to look beyond microRNA cargos and pursue innovative pathways through which dietary exosomes alter metabolism. Here, we highlight potentially fruitful lines of exploration

Genetically Altered Bovine Milk Exosomes (BMEs) Evade Elimination byMurine Bone Marrow-Derived Macrophages (BMDMs)

Objectives: To develop BMEs that evade elimination by BMDMs. Conclusions: The elimination of BMEs UNL1 and UNL2 is significantly reduced compared unmodified BMEs in BMDM cultures. This is of great importance when using BMEs for delivering therapeutics

Intracellular Fate of Bovine Milk Exosomes in Murine Bone Marrow-Derived Macrophages

Milk exosomes (MEs) and their microRNA cargos constitute novel bioactive food compounds, and bovine MEs (BMEs) are being considered for use in drug delivery. The internalization of MEs by macrophages and degradation in lysosomes (in all cells) limit biological activities. Here, we determined whether BMEs internalized by murine bone marrow-derived macrophages (BMDMs) retrofuse with the intralumenal membrane in the multivesicular body (MVB) for subsequent release into the extracellular space or whether BMEs are destined for degradation in lysosomes. Conclusions: The retrofusion of BME-derived intralumenal MVBs and secretion of BME into the extracellular space is quantitatively minor in BMDMs. The majority of BMEs localizes to lysosomes for degradation in BMDMs

Nutrition, Histone Epigenetic Marks, and Disease

The dietary intake of essential nutrients and bioactive food compounds is a process that occurs on a daily basis for the entire life span. Therefore, your diet has a great potential to cause changes in the epigenome. Known histone modifications include acetylation, methylation, biotinylation, poly(ADP-ribosylation), ubiquitination, and sumoylation. Some of these modifications depend directly on dietary nutrients. For other modifications, bioactive dietary compounds may alter the activities of enzymes that establish or remove histone marks, thereby altering the epigenome. This chapter provides an overview of diet-dependent epigenomic marks in histones and their links with human health

Milk-borne small extracellular vesicles: kinetics and mechanisms of transport, distribution, and elimination

Small extracellular vesicles (sEVs) in milk have the qualities desired for delivering therapeutics to diseased tissues. The production of bovine milk sEVs is scalable (1021 annually per cow), and they resist degradation in the gastrointestinal tract. Most cells studied to date internalize milk sEVs by a saturable process that follows Michaelis-Menten kinetics. The bioavailability of oral milk sEVs is approximately 50%. In addition to crossing the intestinal mucosa, milk sEVs also cross barriers such as the placenta and blood-brain barrier, thereby enabling the delivery of therapeutics to hard-to-reach tissues. In time course studies, levels of milk sEVs peaked in the intestinal mucosa, plasma, and urine approximately 6 h and returned to baseline 24 h after oral gavage in mice. In tissues, milk sEV levels peaked 12 h after gavage. Milk sEVs appear to be biologically safe. No cytokine storm was observed when milk sEVs were added to cultures of human peripheral blood mononuclear cells or administered orally to rats. Liver and kidney function and erythropoiesis were not impaired when milk sEVs were administered to rats by oral gavage for up to 15 days. Protocols for loading milk sEVs with therapeutic cargo are available. Currently, the use of milk sEVs (and other nanoparticles) in the delivery of therapeutics is limited by their rapid elimination through internalization by macrophages and lysosomal degradation in target cells. This mini review discusses the current knowledge base of sEV tissue distribution, excretion in feces and urine, internalization by macrophages, and degradation in lysosomes

Nutrition, Histone Epigenetic Marks, and Disease

The dietary intake of essential nutrients and bioactive food compounds is a process that occurs on a daily basis for the entire life span. Therefore, your diet has a great potential to cause changes in the epigenome. Known histone modifications include acetylation, methylation, biotinylation, poly(ADP-ribosylation), ubiquitination, and sumoylation. Some of these modifications depend directly on dietary nutrients. For other modifications, bioactive dietary compounds may alter the activities of enzymes that establish or remove histone marks, thereby altering the epigenome. This chapter provides an overview of diet-dependent epigenomic marks in histones and their links with human health

Holocarboxylase synthetase regulates expression of biotin transporters by chromatin remodeling events at the SMVT locus

The sodium-dependent multivitamin transporter (SMVT) is essential for mediating and regulating biotin entry into mammalian cells. In cells, biotin is covalently linked to histones in a reaction catalyzed by holocarboxylase synthetase (HCS); biotinylation of lysine 12-biotinylated histone H4 (K12Bio H4) causes gene silencing. Here, we propose a novel role for HCS in sensing and regulating levels of biotin in eukaryotic cells. We hypothesized that nuclear translocation of HCS increases in response to biotin supplementation; HCS then biotinylates histone H4 at SMVT promoters, silencing biotin transporter genes. Jurkat lymphoma cells were cultured in media containing 0.025, 0.25, or 10 nmol/l biotin. The nuclear translocation of HCS correlated with biotin concentrations in media; the relative enrichment of both HCS and K12Bio H4 at SMVT promoter 1 (but not promoter 2) increased by 91% in cells cultured in medium containing 10 nmol/l biotin compared with 0.25 nmol/l biotin. This increase of K12Bio H4 at the SMVT promoter decreased SMVT expression by up to 86%. Biotin homeostasis by HCS-dependent chromatin remodeling at the SMVT promoter 1 locus was disrupted in HCS knockdown cells, as evidenced by abnormal chromatin structure (K12Bio H4 abundance) and increased SMVT expression. The findings from this study are consistent with the theory that HCS senses biotin, and that biotin regulates its own cellular uptake by participating in HCS-dependent chromatin remodeling events at the SMVT promoter 1 locus in Jurkat cells.Fil: Gralla, Michael. Universidad de Nebraska - Lincoln; Estados UnidosFil: Camporeale, Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Universidad de Nebraska - Lincoln; Estados UnidosFil: Zempleni, Janos. Universidad de Nebraska - Lincoln; Estados Unido

The polypeptide Syn67 interacts physically with human holocarboxylase synthetase, but is not a target for biotinylation

Holocarboxylase synthetase (HCS) catalyzes the binding of biotin to lysines in carboxylases and histones in two steps. First, HCS catalyzes the synthesis of biotinyl-5′-AMP; second, the biotinyl moiety is ligated to lysine residues. It has been proposed that step two is fairly promiscuous, and that protein biotinylation may occur in the absence of HCS as long as sufficient exogenous biotinyl-5′- AMP is provided. Here, we identified a novel polypeptide (Syn67) with a basic patch of lysines and arginines. Yeast-two-hybrid assays and limited proteolysis assays revealed that both N- and C-termini of HCS interact with Syn67. A potential target lysine in Syn67 was biotinylated by HCS only after arginine-to-glycine substitutions in Syn67 produced a histone-like peptide. We identified a Syn67 docking site near the active pocket of HCS by in silico modeling and site directed mutagenesis. Biotinylation of proteins by HCS is more specific than previously assumed

The polypeptide Syn67 interacts physically with human holocarboxylase synthetase, but is not a target for biotinylation

Holocarboxylase synthetase (HCS) catalyzes the binding of biotin to lysines in carboxylases and histones in two steps. First, HCS catalyzes the synthesis of biotinyl-5′-AMP; second, the biotinyl moiety is ligated to lysine residues. It has been proposed that step two is fairly promiscuous, and that protein biotinylation may occur in the absence of HCS as long as sufficient exogenous biotinyl-5′- AMP is provided. Here, we identified a novel polypeptide (Syn67) with a basic patch of lysines and arginines. Yeast-two-hybrid assays and limited proteolysis assays revealed that both N- and C-termini of HCS interact with Syn67. A potential target lysine in Syn67 was biotinylated by HCS only after arginine-to-glycine substitutions in Syn67 produced a histone-like peptide. We identified a Syn67 docking site near the active pocket of HCS by in silico modeling and site directed mutagenesis. Biotinylation of proteins by HCS is more specific than previously assumed

Off target effects of sulforaphane include the de-repression of long-terminal repeats through histone acetylation events

Sulforaphane is a naturally occurring isothiocyanate in cruciferous vegetables. Sulforaphane inhibits histone deacetylases, leading to the transcriptional activation of genes including tumor suppressor genes. The compound has attracted considerable attention in the chemoprevention of prostate cancer. Here we tested the hypothesis that sulforaphane is not specific for tumor suppressor genes but also activates loci such as long terminal repeats (LTRs), which might impair genome stability. Studies were conducted using chemically pure sulforaphane in primary human IMR-90 fibroblasts and in broccoli sprout feeding studies in healthy adults. Sulforaphane (2.0 μM) caused an increase in LTR transcriptional activity in cultured cells. Consumption of broccoli sprouts (34, 68, or 102g) by human volunteers caused a dose dependent elevation in LTR mRNA in circulating leukocytes, peaking at more than a 10-fold increase. This increase in transcript levels was associated with an increase in histone H3 K9 acetylation marks in LTR 15 in peripheral blood mononuclear cells from subjects consuming sprouts. Collectively, this study suggests that sulforaphane has off-target effects that warrant further investigation when recommending high levels of sulforaphane intake, despite its promising activities in chemoprevention