Structural analysis of the full-length human LRRK2.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are commonly implicated in the pathogenesis of both familial and sporadic Parkinson's disease (PD). LRRK2 regulates critical cellular processes at membranous organelles and forms microtubule-based pathogenic filaments, yet the molecular basis underlying these biological roles of LRRK2 remains largely enigmatic. Here, we determined high-resolution structures of full-length human LRRK2, revealing its architecture and key interdomain scaffolding elements for rationalizing disease-causing mutations. The kinase domain of LRRK2 is captured in an inactive state, a conformation also adopted by the most common PD-associated mutation, LRRK2G2019S. This conformation serves as a framework for structure-guided design of conformational specific inhibitors. We further determined the structure of COR-mediated LRRK2 dimers and found that single-point mutations at the dimer interface abolished pathogenic filamentation in cells. Overall, our study provides mechanistic insights into physiological and pathological roles of LRRK2 and establishes a structural template for future therapeutic intervention in PD.

The protective effect of the mitochondrial-derived peptide MOTS-c on LPS-induced septic cardiomyopathy.

Septic cardiomyopathy is associated with mechanisms such as excessive inflammation, oxidative stress, regulation of calcium homeostasis, endothelial dysfunction, mitochondrial dysfunction, and cardiomyocyte death, and there is no effective treatment at present. MOTS-c is a mitochondria-derived peptide (MDP) encoded by mitochondrial DNA (mtDNA) that protects cells from stresses in an AMPK-dependent manner. In the present study, we aim to explore the protective effect of MOTS-c on lipopolysaccharide (LPS)-induced septic cardiomyopathy. LPS is used to establish a model of septic cardiomyopathy. Our results demonstrate that MOTS-c treatment reduces the mRNA levels of inflammatory cytokines ( IL-1ß, IL-4, IL-6, and TNFα) in cardiomyocytes and the levels of circulating myocardial injury markers, such as CK-MB and TnT, alleviates cardiomyocyte mitochondrial dysfunction and oxidative stress, reduces cardiomyocyte apoptosis, activates cardioprotection-related signaling pathways, including AMPK, AKT, and ERK, and inhibits the inflammation-related signaling pathways JNK and STAT3. However, treatment with the AMPK pathway inhibitor compound C (CC) abolishes the positive effect of MOTS-c on LPS stress. Collectively, our research suggests that MOTS-c may attenuate myocardial injury in septic cardiomyopathy by activating AMPK and provides a new idea for therapeutic strategies in septic cardiomyopathy.

Genome-wide methylomic regulation of multiscale gene networks in Alzheimer's disease.

INTRODUCTION: Recent studies revealed the association of abnormal methylomic changes with Alzheimer's disease (AD) but there is a lack of systematic study of the impact of methylomic alterations over the molecular networks underlying AD. METHODS: We profiled genome-wide methylomic variations in the parahippocampal gyrus from 201 post mortem control, mild cognitive impaired, and AD brains. RESULTS: We identified 270 distinct differentially methylated regions (DMRs) associated with AD. We quantified the impact of these DMRs on each gene and each protein as well as gene and protein co-expression networks. DNA methylation had a profound impact on both AD-associated gene/protein modules and their key regulators. We further integrated the matched multi-omics data to show the impact of DNA methylation on chromatin accessibility, which further modulates gene and protein expression. DISCUSSION: The quantified impact of DNA methylation on gene and protein networks underlying AD identified potential upstream epigenetic regulators of AD. HIGHLIGHTS: A cohort of DNA methylation data in the parahippocampal gyrus was developed from 201 post mortem control, mild cognitive impaired, and Alzheimer's disease (AD) brains. Two hundred seventy distinct differentially methylated regions (DMRs) were found to be associated with AD compared to normal control. A metric was developed to quantify methylation impact on each gene and each protein. DNA methylation was found to have a profound impact on not only the AD-associated gene modules but also key regulators of the gene and protein networks. Key findings were validated in an independent multi-omics cohort in AD. The impact of DNA methylation on chromatin accessibility was also investigated by integrating the matched methylomic, epigenomic, transcriptomic, and proteomic data.

Deep Single-Cell-Type Proteome Profiling of Mouse Brain by Nonsurgical AAV-Mediated Proximity Labeling.

Proteome profiling is a powerful tool in biological and biomedical studies, starting with samples at bulk, single-cell, or single-cell-type levels. Reliable methods for extracting specific cell-type proteomes are in need, especially for the cells (e.g., neurons) that cannot be readily isolated. Here, we present an innovative proximity labeling (PL) strategy for single-cell-type proteomics of mouse brain, in which TurboID (an engineered biotin ligase) is used to label almost all proteins in a specific cell type. This strategy bypasses the requirement of cell isolation and includes five major steps: (i) constructing recombinant adeno-associated viruses (AAVs) to express TurboID driven by cell-type-specific promoters, (ii) delivering the AAV to mouse brains by direct intravenous injection, (iii) enhancing PL labeling by biotin administration, (iv) purifying biotinylated proteins, followed by on-bead protein digestion, and (v) quantitative tandem-mass-tag (TMT) labeling. We first confirmed that TurboID can label a wide range of cellular proteins in human HEK293 cells and optimized the single-cell-type proteomic pipeline. To analyze specific brain cell types, we generated recombinant AAVs to coexpress TurboID and mCherry proteins, driven by neuron- or astrocyte-specific promoters and validated the expected cell expression by coimmunostaining of mCherry and cellular markers. Subsequent biotin purification and TMT analysis identified ∼10,000 unique proteins from a few micrograms of protein samples with excellent reproducibility. Comparative and statistical analyses indicated that these PL proteomes contain cell-type-specific cellular pathways. Although PL was originally developed for studying protein-protein interactions and subcellular proteomes, we extended it to efficiently tag the entire proteomes of specific cell types in the mouse brain using TurboID biotin ligase. This simple, effective in vivo approach should be broadly applicable to single-cell-type proteomics.

Ubiquitination independent of E1 and E2 enzymes by bacterial effectors.

Signalling by ubiquitination regulates virtually every cellular process in eukaryotes. Covalent attachment of ubiquitin to a substrate is catalysed by the E1, E2 and E3 three-enzyme cascade, which links the carboxy terminus of ubiquitin to the ε-amino group of, in most cases, a lysine of the substrate via an isopeptide bond. Given the essential roles of ubiquitination in the regulation of the immune system, it is not surprising that the ubiquitination network is a common target for diverse infectious agents. For example, many bacterial pathogens exploit ubiquitin signalling using virulence factors that function as E3 ligases, deubiquitinases or as enzymes that directly attack ubiquitin. The bacterial pathogen Legionella pneumophila utilizes approximately 300 effectors that modulate diverse host processes to create a permissive niche for its replication in phagocytes. Here we demonstrate that members of the SidE effector family of L. pneumophila ubiquitinate multiple Rab small GTPases associated with the endoplasmic reticulum. Moreover, we show that these proteins are capable of catalysing ubiquitination without the need for the E1 and E2 enzymes. A putative mono-ADP-ribosyltransferase motif critical for the ubiquitination activity is also essential for the role of the SidE family in intracellular bacterial replication in a protozoan host. The E1/E2-independent ubiquitination catalysed by these enzymes is energized by nicotinamide adenine dinucleotide, which activates ubiquitin by the formation of ADP-ribosylated ubiquitin. These results establish that ubiquitination can be catalysed by a single enzyme, the activity of which does not require ATP.

Deep Profiling of Microgram-Scale Proteome by Tandem Mass Tag Mass Spectrometry.

Tandem mass tag (TMT)-based mass spectrometry (MS) enables deep proteomic profiling of more than 10,000 proteins in complex biological samples but requires up to 100 µg protein in starting materials during a standard analysis. Here, we present a streamlined protocol to quantify more than 9000 proteins with 0.5 µg protein per sample by 16-plex TMT coupled with two-dimensional liquid chromatography and tandem mass spectrometry (LC/LC-MS/MS). In this protocol, we optimized multiple conditions to reduce sample loss, including processing each sample in a single tube to minimize surface adsorption, increasing digestion enzymes to shorten proteolysis and function as carriers, eliminating a desalting step between digestion and TMT labeling, and developing miniaturized basic pH LC for prefractionation. By profiling 16 identical human brain tissue samples of Alzheimer's disease (AD), vascular dementia (VaD), and non-dementia controls, we directly compared this new microgram-scale protocol to the standard-scale protocol, quantifying 9116 and 10,869 proteins, respectively. Importantly, bioinformatics analysis indicated that the microgram-scale protocol had adequate sensitivity and reproducibility to detect differentially expressed proteins in disease-related pathways. Thus, this newly developed protocol is of general application for deep proteomics analysis of biological and clinical samples at sub-microgram levels.

27-Plex Tandem Mass Tag Mass Spectrometry for Profiling Brain Proteome in Alzheimer's Disease.

Multiplexed isobaric labeling methods, such as tandem mass tags (TMT), remarkably improve the throughput of quantitative mass spectrometry. Here, we present a 27-plex TMT method coupled with two-dimensional liquid chromatography (LC/LC) for extensive peptide fractionation and high-resolution tandem mass spectrometry (MS/MS) for peptide quantification and then apply the method to profile the complex human brain proteome of Alzheimer's disease (AD). The 27-plex method combines multiplexed capacities of the 11-plex and the 16-plex TMT, as the peptides labeled by the two TMT sets display different mass and hydrophobicity, which can be well separated in LC-MS/MS. We first systematically optimized the protocol for the newly developed 16-plex TMT, including labeling reaction, desalting, and MS conditions, and then directly compared the 11-plex and 16-plex methods by analyzing the same human AD samples. Both methods yielded similar proteome coverage, analyzing >100 000 peptides in >10 000 human proteins. Furthermore, the 11-plex and 16-plex samples were mixed for a 27-plex assay, resulting in more than 8000 protein measurements within the same MS time. The 27-plex results are highly consistent with those of the individual 11-plex and 16-plex TMT analyses. We also used these proteomics data sets to compare the AD brain with the nondementia controls, discovering major AD-related proteins and revealing numerous novel protein alterations enriched in the pathways of amyloidosis, immunity, mitochondrial, and synaptic functions. Overall, our data strongly demonstrate that this new 27-plex strategy is highly feasible for routine large-scale proteomic analysis.

Identification of a Novel Salmonella Type III Effector by Quantitative Secretome Profiling.

Salmonella enterica serovar Typhimurium is arguably one of the most studied bacterial pathogens and successful infection requires the delivery of its virulence factors (effectors) directly into host cells via the type III secretion systems (T3SSs). Central to Salmonella pathogenesis, these effector proteins have been subjected to extensive studies over the years. Nevertheless, whether additional effectors exist remains unclear. Here we report the identification of a novel Salmonella T3SS effector STM1239 (which we renamed SopF) via quantitative secretome profiling. Immunoblotting and ß-lactamase reporter assays confirmed the secretion and translocation of SopF in a T3SS-dependent manner. Moreover, ectopic expression of SopF caused significant toxicity in yeast cells. Importantly, genetic ablation of sopF led to Salmonella strains defective in intracellular replication within macrophages and the mutant were also markedly attenuated in a mouse model of infection. Our study underscores the use of quantitative secretome profiling in identifying novel virulence factors for bacterial pathogens.

Periostin inhibits mechanical stretch-induced apoptosis in osteoblast-like MG-63 cells.

BACKGROUND/PURPOSE: Appropriate mechanical stress plays an important role in regulating the proliferation and differentiation of osteoblasts, whereas high-level mechanical stress may be harmful and compromise cell survival. Periostin, a matricellular protein, is essential in maintaining functional integrity of bone and collagen-rich connective tissue in response to mechanical stress. This study investigated whether or not high-level mechanical stretch induces cell apoptosis and the regulatory role of periostin in mechanical stretch-induced apoptosis in osteoblastic cells. METHODS: Osteoblast-like MG-63 cells were seeded onto Bio-Flex I culture plates and subjected to cyclic mechanical stretching (15% elongation, 0.1 Hz) in a Flexercell tension plus system-5000. The same process was applied to cells pre-treated with exogenous human recombinant periostin before mechanical stretching. We used a chromatin condensation and membrane permeability dead cell apoptosis kit to evaluate the stretch-induced cell responses. Expression of caspase-3 and cPARP was examined by immunofluorescent stain and flow cytometry. RESULTS: The expression of periostin in MG-63 cells is involved in the TGF-ß signaling pathway. High-level cyclic mechanical stretch induced apoptotic responses in MG-63 osteoblastic cells. The percentages of apoptotic cells and cells expressing cPARP protein increased in the groups of cells subjected to mechanical stretch, but these responses were absent in the presence of exogenous periostin. CONCLUSION: Our study revealed that high-level mechanical stretch induces apoptotic cell death, and that periostin plays a protective role against mechanical stretch-induced apoptosis in osteoblastic cells.

Quantitative analysis of Shigella flexneri protein expression under acid stress.

As an important foodborne pathogen, Shigella flexneri can cause widespread enteric infection with bacteria as few as hundreds. This is, at least in part, attributed to its robust anti-acid strategies because passage through the highly acidic human digestive tract is a prerequisite for successful bacterial infection. Nevertheless, our understanding of these mechanisms and the impact of acid stress on Shigella protein expression still remains largely incomplete. Herein we conducted a proteomic survey of Shigella spp. under acid stress. Out of 1754 protein identifications, we found 131 altered proteins, most of which were down-regulated, including virulence factors and cell envelope proteins. Rather, many metabolic enzymes and pyrimidine/amino acid biosynthesis proteins were up-regulated. In addition to induction of many known anti-acid systems, we also found marked increase of 2-oxoglutarate dehydrogenase (SucAB), a metabolic enzyme in the tricarboxylic acid cycle. Importantly, overproduction of this enzyme significantly enhanced Shigella acid resistance and hence SucAB-mediated metabolic pathways may represent novel anti-acid strategies.

Quantitative proteomic analysis of host epithelial cells infected by Salmonella enterica serovar Typhimurium.

Systems-level analyses have the capability to offer new insight into host-pathogen interactions on the molecular level. Using Salmonella infection of host epithelial cells as a model system, we previously analyzed intracellular bacterial proteome as a window into pathogens' adaptations to their host environment [Infect. Immun. 2015; J. Proteome Res. 2017]. Herein we extended our efforts to quantitatively examine protein expression of host cells during infection. In total, we identified more than 5000 proteins with 194 differentially regulated proteins upon bacterial infection. Notably, we found marked induction of host integrin signaling and glycolytic pathways. Intriguingly, up-regulation of host glucose metabolism concurred with increased utilization of glycolysis by intracellular Salmonella during infection. In addition to immunoblotting assays, we also verified the up-regulation of PARP1 in the host nucleus by selected reaction monitoring and immunofluorescence studies. Furthermore, we provide evidence that PARP1 elevation is likely specific to Salmonella infection and independent of one of the bacterial type III secretion systems. Our work demonstrates that unbiased high-throughput proteomics can be used as a powerful approach to provide new perspectives on host-pathogen interactions.

Quantitative Proteomics Charts the Landscape of Salmonella Carbon Metabolism within Host Epithelial Cells.

We performed a proteomic survey of Salmonella enterica serovar Typhimurium during infection of host epithelial cells. Our data reveal substantial metabolic reshuffling of Salmonella in the host in addition to severe degeneration of bacterial flagella and chemotaxis systems. The large-scale quantitative data allowed us to chart an overview of intracellular Salmonella carbon metabolism. Notably, we found preferential utilization of glycolysis, the pentose phosphate pathway, mixed acid fermentation, and nucleotide metabolism. In contrast, the tricarboxylic acid (TCA) cycle and aerobic and anaerobic respiration pathways were largely repressed. Furthermore, inactivation of glycolysis and purine biosynthesis led to severe growth defects, indicating important roles in intracellular Salmonella replication. In summary, we exploited quantitative proteomics for rational design of follow-up genetic studies and identified pathways important for bacterial fitness within host cells.

Acetylation of p53 Protein at Lysine 120 Up-regulates Apaf-1 Protein and Sensitizes the Mitochondrial Apoptotic Pathway.

The p53 tumor suppressor controls cell growth, metabolism, and death by regulating the transcription of various target genes. The target-specific transcriptional activity of p53 is highly regulated. Here we demonstrate that acetylation of p53 at Lys-120 up-regulates its transcriptional activity toward Apaf-1, a core component in the mitochondrial apoptotic pathway, and thus sensitizes caspase activation and apoptosis. We found that histone deacetylase (HDAC) inhibitors, including butyrate, augment Lys-120 acetylation of p53 and thus Apaf-1 expression by inhibiting HDAC1. In p53-null cells, transfection of wild-type but not K120R mutant p53 can restore the p53-dependent sensitivity to butyrate. Strikingly, transfection of acetylation-mimicking K120Q mutant p53 is sufficient to up-regulates Apaf-1 in a manner independent of butyrate treatment. Therefore, HDAC inhibitors can induce p53 acetylation at lysine 120, which in turn enhances mitochondrion-mediated apoptosis through transcriptional up-regulation of Apaf-1.

DNA Dendrimer-Streptavidin Nanocomplex: an Efficient Signal Amplifier for Construction of Biosensing Platforms.

We develop a DNA dendrimer-streptavidin (SA) nanocomplex as a novel signal amplifier to create biosensing platforms for disease-related species. The DNA dendrimer-SA nanocomplex is fabricated by cross-linking the nonlinear hybridization chain reaction based DNA dendrimer with the SA-coupled linker DNA and possesses multiple sticky ends, a high molecular weight, and a hyperbranched nanostructure with large numbers of DNA duplexes. Taking advantage of the DNA dendrimer-SA nanocomplex and a label-free quartz crystal microbalance (QCM) technology, we first construct a mass-sensitive QCM biosensing platform for nucleic acids, which displays high selectivity and sensitivity, with a detection limit of 0.062 nM KRAS gene fragment. Then we present a fluorescent sensing strategy toward HeLa cells by functionalizing the DNA dendrimer-SA nanocomplex using the sgc8 aptamer and the SYBR Green I intercalating dye. The spiked recoveries of targets in physiological media are greater than 90%, demonstrating potential application of created biosensing platforms in clinical diagnosis. This work expands the rule set of designing DNA nanomaterials for development of biosensing strategies, and provides universal platforms for detecting disease-related species through simply altering the related capture and reporter DNA sequences.

Biosensor-assisted transcriptional regulator engineering for Methylobacterium extorquens AM1 to improve mevalonate synthesis by increasing the acetyl-CoA supply.

Acetyl-CoA is not only an important intermediate metabolite for cells but also a significant precursor for production of industrially interesting metabolites. Methylobacterium extorquens AM1, a model strain of methylotrophic cell factories using methanol as carbon source, is of interest because it produces abundant coenzyme A compounds capable of directing to synthesis of different useful compounds from methanol. However, acetyl-CoA is not always efficiently accumulated in M. extorquens AM1, as it is located in the center of three cyclic central metabolic pathways. Here we successfully demonstrated a strategy for sensor-assisted transcriptional regulator engineering (SATRE) to control metabolic flux re-distribution to increase acetyl-CoA flux from methanol for mevalonate production in M. extorquens AM1 with introduction of mevalonate synthesis pathway. A mevalonate biosensor was constructed and we succeeded in isolating a mutated strain (Q49) with a 60% increase in mevalonate concentration (an acetyl-CoA-derived product) following sensor-based high-throughput screening of a QscR transcriptional regulator library. The mutated QscR-49 regulator (Q8*,T61S,N72Y,E160V) lost an N-terminal α-helix and underwent a change in the secondary structure of the RD-I domain at the C terminus, two regions that are related to its interaction with DNA. 13C labeling analysis revealed that acetyl-CoA flux was improved by 7% and transcriptional analysis revealed that QscR had global effects and that two key points, NADPH generation and fumC overexpression, might contribute to the carbon flux re-distribution. A fed-batch fermentation in a 5-L bioreactor for QscR-49 mutant yielded a mevalonate concentration of 2.67g/L, which was equivalent to an overall yield of 0.055mol acetyl-CoA/mol methanol, the highest yield among engineered strains of M. extorquens AM1. This work was the first attempt to regulate M. extorquens AM1 on transcriptional level and provided molecular insights into the mechanism of carbon flux regulation.

Proteomic Analyses of Intracellular Salmonella enterica Serovar Typhimurium Reveal Extensive Bacterial Adaptations to Infected Host Epithelial Cells.

Salmonella species can gain access into nonphagocytic cells, where the bacterium proliferates in a unique membrane-bounded compartment. In order to reveal bacterial adaptations to their intracellular niche, here we conducted the first comprehensive proteomic survey of Salmonella isolated from infected epithelial cells. Among ∼ 3,300 identified bacterial proteins, we found that about 100 proteins were significantly altered at the onset of Salmonella intracellular replication. In addition to substantially increased iron-uptake capacities, bacterial high-affinity manganese and zinc transporters were also upregulated, suggesting an overall limitation of metal ions in host epithelial cells. We also found that Salmonella induced multiple phosphate utilization pathways. Furthermore, our data suggested upregulation of the two-component PhoPQ system as well as of many downstream virulence factors under its regulation. Our survey also revealed that intracellular Salmonella has increased needs for certain amino acids and biotin. In contrast, Salmonella downregulated glycerol and maltose utilization as well as chemotaxis pathways.

Selective CK1α degraders exert antiproliferative activity against a broad range of human cancer cell lines.

Molecular-glue degraders are small molecules that induce a specific interaction between an E3 ligase and a target protein, resulting in the target proteolysis. The discovery of molecular glue degraders currently relies mostly on screening approaches. Here, we describe screening of a library of cereblon (CRBN) ligands against a panel of patient-derived cancer cell lines, leading to the discovery of SJ7095, a potent degrader of CK1α, IKZF1 and IKZF3 proteins. Through a structure-informed exploration of structure activity relationship (SAR) around this small molecule we develop SJ3149, a selective and potent degrader of CK1α protein in vitro and in vivo. The structure of SJ3149 co-crystalized in complex with CK1α + CRBN + DDB1 provides a rationale for the improved degradation properties of this compound. In a panel of 115 cancer cell lines SJ3149 displays a broad antiproliferative activity profile, which shows statistically significant correlation with MDM2 inhibitor Nutlin-3a. These findings suggest potential utility of selective CK1α degraders for treatment of hematological cancers and solid tumors.

Genome-wide analyses of lung cancer after single high-dose radiation at five time points (2, 6, 12, 24, and 48 h).

Background: An increasing number of clinicians are experimenting with high-dose radiation. This study focuses on the genomic effects of high-dose single-shot radiotherapy and aims to provide a dynamic map for non-small cell lung cancer (NSCLC). Methods: We used whole-transcriptome sequencing to understand the evolution at molecular levels in A549 and H1299 exposed to 10 Gy X-rays at different times (2, 6, 12, 24, and 48 h) in comparison with the no radiation group. Ingenuity pathway analysis, ceRNA analysis, enrichment analysis, and cell cycle experiments are performed for molecular analyses and function analyses. Results: Whole-transcriptome sequencing of NSCLC showed a significant dynamic change after radiotherapy within 48 h. MiR-219-1-3p and miR-221-3p, miR-503-5p, hsa-miR-455-5p, hsa-miR-29-3p, and hsa-miR-339-5p were in the core of the ceRNA related to time change. GO and KEGG analyses of the top 30 mRNA included DNA repair, autophagy, apoptosis, and ferroptosis pathways. Regulation of the cell cycle-related transcription factor E2F1 might have a key role in the early stage of radiotherapy (2.6 h) and in the later stage of autophagy (24 and 48 h). Functions involving different genes/proteins over multiple periods implied a dose of 10 Gy was related to the kidney and liver pathway. Radiation-induced cell cycle arrest at the G2/M phase was evident at 24 h. We also observed the increased expression of CCNB1 at 24 h in PCR and WB experiments. Conclusion: Our transcriptomic and experimental analyses showed a dynamic change after radiation therapy in 48 h and highlighted the key molecules and pathways in NSCLC after high-dose single-shot radiotherapy.

Phosphoribosyl-linked serine ubiquitination of USP14 by the SidE family effectors of Legionella excludes p62 from the bacterial phagosome.

Xenophagy is an evolutionarily conserved host defensive mechanism to eliminate invading microorganisms through autophagic machinery. The intracellular bacterial pathogen Legionella pneumophila can avoid clearance by the xenophagy pathway via the actions of multiple Dot/Icm effector proteins. Previous studies have shown that p62, an adaptor protein involved in xenophagy signaling, is excluded from Legionella-containing vacuoles (LCVs). Such defects are attributed to the multifunctional SidE family effectors (SidEs) that exhibit classic deubiquitinase (DUB) and phosphoribosyl ubiquitination (PR-ubiquitination) activities, yet the mechanism remains elusive. In the present study, we demonstrate that the host DUB USP14 is PR-ubiquitinated by SidEs at multiple serine residues, which impairs its DUB activity and its interactions with p62. The exclusion of p62 from the bacterial phagosome requires the ubiquitin ligase but not the DUB activity of SidEs. These results reveal that PR-ubiquitination of USP14 by SidEs contributes to the evasion of xenophagic clearance by L. pneumophila.

Comprehensive characterization of human brain-derived extracellular vesicles using multiple isolation methods: Implications for diagnostic and therapeutic applications.

Extracellular vesicles (EVs) have emerged as critical mediators of intercellular communication and promising biomarkers and therapeutics in the central nervous system (CNS). Human brain-derived EVs (BDEVs) provide a comprehensive snapshot of physiological changes in the brain's environment, however, the isolation of BDEVs and the comparison of different methods for this purpose have not been fully investigated. In this study, we compared the yield, morphology, subtypes and protein cargo composition of EVs isolated from the temporal cortex of aged human brains using three established separation methods: size-exclusion chromatography (SEC), phosphatidylserine affinity capture (MagE) and sucrose gradient ultracentrifugation (SG-UC). Our results showed that SG-UC method provided the highest yield and collected larger EVs compared to SEC and MagE methods as assessed by transmission electron microscopy and nanoparticle tracking analysis (NTA). Quantitative tandem mass-tag (TMT) mass spectrometry analysis of EV samples from three different isolation methods identified a total of 1158 proteins, with SG-UC showing the best enrichment of common EV proteins with less contamination of non-EV proteins. In addition, SG-UC samples were enriched in proteins associated with ATP activity and CNS maintenance, and were abundant in neuronal and oligodendrocytic molecules. In contrast, MagE samples were more enriched in molecules related to lipoproteins, cell-substrate junction and microglia, whereas SEC samples were highly enriched in molecules related to extracellular matrix, Alzheimer's disease and astrocytes. Finally, we validated the proteomic results by performing single-particle analysis using the super-resolution microscopy and flow cytometry. Overall, our findings demonstrate the differences in yield, size, enrichment of EV cargo molecules and single EV assay by different isolation methods, suggesting that the choice of isolation method will have significant impact on the downstream analysis and protein discovery.