Preliminary Characterization of Extracellular Vesicles From Auditory HEI-OC1 Cells.

ObjectivesIsolate, purify, and characterize extracellular vesicles (EVs) obtained from auditory HEI-OC1 cells, and evaluate their suitability for intracochlear transport and delivery of pharmacological drugs and/or pro-resolution mediators of acute inflammatory processes.MethodsHEI-OC1 EVs were isolated and purified using the exoEasy Maxi Kit, and their size was evaluated by nanoparticle tracking techniques. Bottom-up proteomics of the EVs, either freshly obtained or stored for up to 4 months at -20°C, was performed by LC-ESI-MS/MS. LC-ESI-MS/MS-MRM was used to measure the loading of dexamethasone inside EVs following co-incubation at room temperature for 1 hour with and without 5 minutes sonication.ResultsRoutinely, we were able to obtain purified fractions of >2 × 109 EVs/mL, with diameters varying between 50 and 800 nm. Bottom-up proteomics showed that among the most abundant EVs proteins, 19.2% were cytoplasmic, 17.2% were membrane localized, 12.3% were cytosolic, and 14.6% were nucleolar. No significant differences between fresh and stored EVs were detected. Importantly, co-incubation of HEI-OC1 EVs (1 × 108 EVs/mL) with dexamethasone (10 mM) resulted in the incorporation of 10.1 ± 1.9 nM dexamethasone per milliliter of EVs suspension.ConclusionsAltogether, the results suggest that EVs from HEI-OC1 cells could be advantageously used as biological nanocarriers for the delivery of specific molecules and pharmacological drugs into the inner ear

Anthrax lethal toxin induced lysosomal membrane permeabilization and cytosolic cathepsin release is Nlrp1b/Nalp1b-dependent.

NOD-like receptors (NLRs) are a group of cytoplasmic molecules that recognize microbial invasion or 'danger signals'. Activation of NLRs can induce rapid caspase-1 dependent cell death termed pyroptosis, or a caspase-1 independent cell death termed pyronecrosis. Bacillus anthracis lethal toxin (LT), is recognized by a subset of alleles of the NLR protein Nlrp1b, resulting in pyroptotic cell death of macrophages and dendritic cells. Here we show that LT induces lysosomal membrane permeabilization (LMP). The presentation of LMP requires expression of an LT-responsive allele of Nlrp1b, and is blocked by proteasome inhibitors and heat shock, both of which prevent LT-mediated pyroptosis. Further the lysosomal protease cathepsin B is released into the cell cytosol and cathepsin inhibitors block LT-mediated cell death. These data reveal a role for lysosomal membrane permeabilization in the cellular response to bacterial pathogens and demonstrate a shared requirement for cytosolic relocalization of cathepsins in pyroptosis and pyronecrosis

Extracellular Vesicles From Auditory Cells as Nanocarriers for Anti-inflammatory Drugs and Pro-resolving Mediators.

Drug- and noise-related hearing loss are both associated with inflammatory responses in the inner ear. We propose that intracochlear delivery of a combination of pro-resolving mediators, specialized proteins and lipids that accelerate the return to homeostasis by modifying the immune response rather than by inhibiting inflammation, might have a profound effect on the prevention of sensorineural hearing loss. However, intracochlear delivery of such agents requires a reliable and effective method to convey them, fully active, directly to the target cells. The present study provides evidence that extracellular vesicles (EVs) from auditory HEI-OC1 cells may incorporate significant quantities of anti-inflammatory drugs, pro-resolving mediators and their polyunsaturated fatty acid precursors as cargo, and potentially could work as carriers for their intracochlear delivery. EVs generated by HEI-OC1 cells were divided by size into two fractions, small (≤150 nm diameter) and large (>150 nm diameter), and loaded with aspirin, lipoxin A4, resolvin D1, and the polyunsaturated fatty acids (PUFA) arachidonic, eicosapentaenoic, docosahexanoic, and linoleic. Bottom-up proteomics revealed a differential distribution of selected proteins between small and large vesicles. Only 17.4% of these proteins were present in both fractions, whereas 61.5% were unique to smaller vesicles and only 3.7% were exclusively found in the larger ones. Importantly, the pro-resolving protein mediators Annexin A1 and Galectins 1 and 3 were only detected in small vesicles. Lipidomic studies, on the other hand, showed that small vesicles contained higher levels of eicosanoids than large ones and, although all of them incorporated the drugs and molecules investigated, small vesicles were more efficiently loaded with PUFA and the large ones with aspirin, LXA4 and resolvin D1. Importantly, our data indicate that the vesicles contain all necessary enzymatic components for the de novo generation of eicosanoids from fatty acid precursors, including pro-inflammatory agents, suggesting that their cargo should be carefully tailored to avoid interference with their therapeutic purpose. Altogether, these results support the idea that both small and large EVs from auditory HEI-OC1 cells could be used as nanocarriers for anti-inflammatory drugs and pro-resolving mediators

The Structure and Interactions of Periplasmic Domains of Crucial MmpL Membrane Proteins from Mycobacterium tuberculosis

SummaryMycobacterium tuberculosis mycobacterial membrane protein large (MmpL) proteins are important in substrate transport across the inner membrane. Here, we show that MmpL proteins are classified into two phylogenetic clusters, where MmpL cluster II contains three soluble domains (D1, D2, and D3) and has two full-length members, MmpL3 and MmpL11. Significantly, MmpL3 is currently the most druggable M. tuberculosis target. We have solved the 2.4-Å MmpL11-D2 crystal structure, revealing structural homology to periplasmic porter subdomains of RND (multidrug) transporters. The resulting predicted cluster II MmpL membrane topology has D1 and D2 residing, and possibly interacting, within the periplasm. Crosslinking and biolayer interferometry experiments confirm that cluster II D1 and D2 bind with weak affinities, and guided D1-D2 heterodimeric model assemblies. The predicted full-length MmpL3 and MmpL11 structural models reveal key substrate binding and transport residues, and may serve as templates to set the stage for in silico anti-tuberculosis drug development

Amyloid seeding of transthyretin by ex vivo cardiac fibrils and its inhibition

Each of the 30 human amyloid diseases is associated with the aggregation of a particular precursor protein into amyloid fibrils. In transthyretin amyloidosis (ATTR), mutant or wild-type forms of the serum carrier protein transthyretin (TTR), synthesized and secreted by the liver, convert to amyloid fibrils deposited in the heart and other organs. The current standard of care for hereditary ATTR is liver transplantation, which replaces the mutant TTR gene with the wild-type gene. However, the procedure is often followed by cardiac deposition of wild-type TTR secreted by the new liver. Here we find that amyloid fibrils extracted from autopsied and explanted hearts of ATTR patients robustly seed wild-type TTR into amyloid fibrils in vitro. Cardiac-derived ATTR seeds can accelerate fibril formation of wild-type and monomeric TTR at acidic pH and under physiological conditions, respectively. We show that this seeding is inhibited by peptides designed to complement structures of TTR fibrils. These inhibitors cap fibril growth, suggesting an approach for halting progression of ATTR

Proteomics and Mass Spectrometry for Cancer Biomarker Discovery

Proteomics is a rapidly advancing field not only in the field of biology but also in translational cancer research. In recent years, mass spectrometry and associated technologies have been explored to identify proteins or a set of proteins specific to a given disease, for the purpose of disease detection and diagnosis. Such biomarkers are being investigated in samples including cells, tissues, serum/plasma, and other types of body fluids. When sufficiently refined, proteomic technologies may pave the way for early detection of cancer or individualized therapy for cancer. Mass spectrometry approaches coupled with bioinformatic tools are being developed for biomarker discovery and validation. Understanding basic concepts and application of such technology by investigators in the field may accelerate the clinical application of protein biomarkers in disease management

Hydrophobic Proteome Analysis of Triple Negative and Hormone-Receptor-Positive-Her2-Negative Breast Cancer by Mass Spectrometer

It is widely believed that discovery of specific, sensitive, and reliable tumor biomarkers can improve the treatment of cancer. Currently, there are no obvious targets that can be used in treating triple-negative breast cancer (TNBC). To better understand TNBC and find potential biomarkers for targeted treatment, we combined a novel hydrophobic fractionation protocol with mass spectrometry LTQ-orbitrap to explore and compare the hydrophobic sub-proteome of TNBC with another subtype of breast cancer, hormone-receptor-positive-Her2-negative breast cancer (non-TNBC). Hydrophobic sub-proteome of breast cancer is rich in membrane proteins. Hundreds of proteins with various defined key cellular functions were identified from TNBC and non-TNBC tumors. In this study, protein profiles of TNBC and non-TNBC were systematically examined, compared, and validated. We have found that nine keratins are down-regulated and several heat shock proteins are up-regulated in TNBC tissues. Our study may provide insights of molecules that are responsible for the aggressiveness of TNBC. The initial results obtained using a combination of hydrophobic fractionation and nano-LC mass spectrometry analysis of these proteins appear promising in the discovery of potential cancer biomarkers and bio-signatures. When sufficiently refined, this approach may prove useful in improving breast cancer treatment

Comparative Proteomic Analysis of Differentially Expressed Proteins in the Urine of Reservoir Hosts of Leptospirosis

Rattus norvegicus is a natural reservoir host for pathogenic species of Leptospira. Experimentally infected rats remain clinically normal, yet persistently excrete large numbers of leptospires from colonized renal tubules via urine, despite a specific host immune response. Whilst persistent renal colonization and shedding is facilitated in part by differential antigen expression by leptospires to evade host immune responses, there is limited understanding of kidney and urinary proteins expressed by the host that facilitates such biological equilibrium. Urine pellets were collected from experimentally infected rats shedding leptospires and compared to urine from non-infected controls spiked with in vitro cultivated leptospires for analysis by 2-D DIGE. Differentially expressed host proteins include membrane metallo endopeptidase, napsin A aspartic peptidase, vacuolar H+ATPase, kidney aminopeptidase and immunoglobulin G and A. Loa22, a virulence factor of Leptospira, as well as the GroEL, were increased in leptospires excreted in urine compared to in vitro cultivated leptospires. Urinary IgG from infected rats was specific for leptospires. Results confirm differential protein expression by both host and pathogen during chronic disease and include markers of kidney function and immunoglobulin which are potential biomarkers of infection

Photoaffinity labeling with cholesterol analogues precisely maps a cholesterol-binding site in voltage-dependent anion channel-1

Voltage-dependent anion channel-1 (VDAC1) is a highly regulated β-barrel membrane protein that mediates transport of ions and metabolites between the mitochondria and cytosol of the cell. VDAC1 co-purifies with cholesterol and is functionally regulated by cholesterol, among other endogenous lipids. Molecular modeling studies based on NMR observations have suggested five cholesterol-binding sites in VDAC1, but direct experimental evidence for these sites is lacking. Here, to determine the sites of cholesterol binding, we photolabeled purified mouse VDAC1 (mVDAC1) with photoactivatable cholesterol analogues and analyzed the photolabeled sites with both top-down mass spectrometry (MS), and bottom-up MS paired with a clickable, stable isotope-labeled tag, FLI-tag. Using cholesterol analogues with a diazirine in either the 7 position of the steroid ring (LKM38) or the aliphatic tail (KK174), we mapped a binding pocket in mVDAC1 localized to Thr83 and Glu73, respectively. When Glu73 was mutated to a glutamine, KK174 no longer photolabeled this residue, but instead labeled the nearby Tyr62 within this same binding pocket. The combination of analytical strategies employed in this work permits detailed molecular mapping of a cholesterol-binding site in a protein, including an orientation of the sterol within the site. Our work raises the interesting possibility that cholesterol-mediated regulation of VDAC1 may be facilitated through a specific binding site at the functionally important Glu73 residue