Cargo Proteins Research Articles

Small extracellular vesicles' enrichment from biological fluids using an acoustic trap.

Small extracellular vesicles (sEVs), a form of extracellular vesicles, are lipid bilayered structures released by all cells. Large-scale studies on sEVs from clinical samples are necessary, but a major obstacle is the lack of rapid, reproducible, efficient, and low-cost methods to enrich sEVs. Acoustic microfluidics have the advantage of being label-free and biocompatible, which have been reported to successfully enrich sEVs. In this paper, we present a highly efficient acoustic microfluidic trap that can offer low and large volume compatible ways of enriching sEVs from biological fluids by flexible structure design. It uses the idea of pre-loading larger seed particles in the acoustic trap to enable sub-micron particle capturing. The microfluidic chip is actuated using a piezoelectric plate transducer attached to a silicon-glass bonding plate with circular cavities. Each cavity works as a resonant unit, excited at the frequency of both the half wave resonance in the main plane and inverted quarter wave resonance in the depth direction, which has the ability to strongly trap seed particles at the center, thereby improving the subsequent nanoparticle capture efficiency. Mean trapping efficiencies of 35.62% and 64.27% were obtained using 60 nm and 100 nm nanobeads, respectively. By the use of this technology, we have successfully enriched sEVs from cell culture conditioned media and blood plasma at a flow rate of 10 μL min-1. The isolated sEV subpopulations are characterized by NTA and TEM, and their protein cargo is determined by WB. This acoustic trapping chip provides a rapid and robust method to enrich sEVs from biofluids with high reproducibility and sufficient quantities. Therefore, it can serve as a new tool for biological and clinical research such as cancer diagnosis and drug delivery.

Extracellular vesicle biogenesis of three-dimensional human pluripotent stem cells in a novel Vertical-Wheel bioreactor.

Extracellular vesicles (EVs) secreted by human-induced pluripotent stem cells (hiPSCs) have great potential as cell-free therapies in various diseases, including prevention of blood-brain barrier senescence and stroke. However, there are still challenges in pre-clinical and clinical use of hiPSC-EVs due to the need for large-scale production of a large quantity. Vertical-Wheel bioreactors (VWBRs) have design features that allow the biomanufacturing of hiPSC-EVs using a scalable aggregate or microcarrier-based culture system under low shear stress. EV secretion by undifferentiated hiPSCs expanded as 3-D aggregates and on Synthemax II microcarriers in VWBRs were investigated. Additionally, two types of EV collection media, mTeSR and HBM, were compared. The hiPSCs were characterized by metabolite and transcriptome analysis as well as EV biogenesis markers. Protein and microRNA cargo were analysed by proteomics and microRNA-seq, respectively. The in vitro functional assays of microglia stimulation and proliferation were conducted. HiPSCs expanded as 3-D aggregates and on microcarriers had comparable cell number, while microcarrier culture had higher glucose consumption, higher glycolysis and lower autophagy gene expression based on mRNA-seq. The microcarrier cultures had at least 17-23 fold higher EV secretion, and EV collection in mTeSR had 2.7-3.7 fold higher yield than HBM medium. Microcarrier culture with mTeSR EV collection had a smaller EV size than other groups, and the cargo was enriched with proteins (proteomics) and miRNAs (microRNA-seq) reducing apoptosis and promoting cell proliferation (e.g. Wnt-related pathways). hiPSC-EVs demonstrated the ability of stimulating proliferation and M2 polarization of microglia in vitro. HiPSC expansion on microcarriers produces much higher yields of EVs than hiPSC aggregates in VWBRs. EV collection in mTeSR increases yield compared to HBM. The biomanufactured EVs from microcarrier culture in mTeSR have exosomal characteristics and are functional in microglia stimulation, which paves the ways for future in vivo anti-aging study.

New Perspectives on Crosstalks Between Bacterial Regulatory RNAs from Outer Membrane Vesicles and Eukaryotic Cells.

Regulatory small RNAs (sRNAs) help the bacteria to survive harsh environmental conditions by posttranscriptional regulation of genes involved in various biological pathways including stress responses, homeostasis, and virulence. These sRNAs can be found carried by different membrane-bound vesicles like extracellular vesicles (EVs), membrane vesicles (MVs), or outer membrane vesicles (OMVs). OMVs provide myriad functions in bacterial cells including carrying a cargo of proteins, lipids, and nucleic acids including sRNAs. A few interesting studies have shown that these sRNAs can be transported to the host cell by membrane vesicles and can regulate the host immune system. Although there is evidence that sRNAs can be exported to host cells and sometimes can even cross the blood-brain barrier, the exact mechanism is still unknown. In this review, we investigated the new techniques implemented in various studies, to elucidate the crosstalks between bacterial cells and human immune systems by membrane vesicles carrying bacterial regulatory sRNAs.

Target-selective cytosolic delivery of cargo proteins using the VHH-presented OLE-ZIP capsules

In the pursuit of a new generation of protein pharmaceuticals, the efficient delivery of these therapeutics into cells stands out as a crucial challenge.

Structural Modeling of T9SS Outer Membrane Proteins and Their Complexes.

The type 9 secretion system (T9SS) is a recently discovered machinery that both transports cargo proteins across the Gram-negative bacterial outer membrane and attaches them to lipopolysaccharides on the extracellular surface. Outer membrane proteins (OMPs) are key components of the T9SS and are involved in both steps. In this chapter, we describe a method for the in silico modeling of T9SS OMPs and their complexes, and model validation. This is useful when the production of recombinant OMPs is difficult, and these protocols can also be applied to OMP complexes outside of the T9SS.

Conventional and Unconventional Protein Secretion in Yeast and Animal Cells.

Protein secretion mediated by the secretory transport pathway is a sophisticated and highly regulated cellular process in eukaryotic cells. In the conventional secretory transport pathway, newly synthesized proteins pass through several endomembrane compartments to reach their destinations. This transport occurs via small, membrane-enclosed vesicles. To ensure the fidelity of trafficking, eukaryotic cells employ elaborate molecular machinery to accurately sort newly synthesized proteins into specific transport vesicles and precisely deliver them to respective acceptor compartments. Leaderless cargo proteins, lacking a signal peptide, follow an unconventional secretory pathway. This review encompasses the molecular machinery regulating both conventional and unconventional protein secretion in yeast and animal cells.

Analysis of Bacterial Extracellular Vesicles by Immunogold Transmission Electron Microscopy.

Bacterial extracellular vesicles (BEVs) have emerged as mediators of transkingdom communication with numerous potential biotechnological applications. As such, investigation of BEV's protein composition holds promise to uncover new biological mechanisms, such as in microbiome-host communication or pathogen infection. Additionally, bioengineering of BEV protein composition can enhance their therapeutic potential. However, accurate assessment of BEV protein cargo is limited by their nanometer size, which precludes light microscopy imaging, as well as by co-isolation of protein impurities during separation processes. A solution to these challenges is found in immunogold transmission electron microscopy (TEM), which combines antibody-based labeling with direct visualization of BEVs. Several challenges are commonly encountered during immunogold TEM analysis of BEVs, most notably inefficient antibody labeling and poor contrast. Here, we present an optimized protocol for immunogold TEM analysis of BEVs that overcomes such challenges.

Tumor necrosis factor-alpha as a modulator of secretory activity and protein cargo of extracellular vesicles in mesenchymal stromal cells

Tumor necrosis factor-alpha as a modulator of secretory activity and protein cargo of extracellular vesicles in mesenchymal stromal cells

Filamentous-Actin-Mimicking Nanoplatform for Enhanced Cytosolic Protein Delivery.

Despite the potential of protein therapeutics, the cytosolic delivery of proteins with high efficiency and bioactivity remains a significant challenge owing to exocytosis and lysosomal degradation after endocytosis. Therefore, it is important to develop a safe and efficient strategy to bypass endocytosis. Inspired by the extraordinary capability of filamentous-actin (F-actin) to promote cell membrane fusion, a cyanine dye assembly-containing nanoplatform mimicking the structure of natural F-actin is developed. The nanoplatform exhibits fast membrane fusion to cell membrane mimics and thus enters live cells through membrane fusion and bypasses endocytosis. Moreover, it is found to efficiently deliver protein cargos into live cells and quickly release them into the cytosol, leading to high protein cargo transfection efficiency and bioactivity. The nanoplatform also results in the superior inhibition of tumor cells when loaded with anti-tumor proteins. These results demonstrate that this fusogenic nanoplatform can be valuable for cytosolic protein delivery and tumor treatment.

Gestational age at birth influences protein and RNA content in human milk extracellular vesicles

AbstractHuman milk extracellular vesicles (HM EVs) are proposed to protect against disease development in infants. This protection could in part be facilitated by the bioactive EV cargo of proteins and RNA. Notably, mothers birth infants of different gestational ages with unique needs, wherein the EV cargo of HM may diverge. We collected HM from lactating mothers within two weeks of a term or preterm birth. Following purification of EVs, proteins and mRNA were extracted for proteomics and sequencing analyses, respectively. Over 2000 protein groups were identified, and over 8000 genes were quantified. The total number of proteins and mRNA did not differ significantly between the two conditions, while functional bioinformatics of differentially expressed cargo indicated enrichment in immunoregulatory cargo for preterm HM EVs. In term HM EVs, significantly upregulated cargo was enriched in metabolism‐related functions. Based on gene expression signatures from HM‐contained single cell sequencing data, we proposed that a larger portion of preterm HM EVs are secreted by immune cells, whereas term HM EVs contain more signatures of lactocyte epithelial cells. Proposed differences in EV cargo could indicate variation in mother's milk based on infants’ gestational age and provide basis for further functional characterisation.

Self-Assembly of Epitope-Tagged Proteins and Antibodies for Delivering Biologics to Antigen Presenting Cells.

Inspired by the immune system's own strategy for macrophage activation, we describe here a simple self-assembly strategy for generating artificial immune complexes. The built-in recognition domains in the antibody, viz. the Fab and Fc domains, are judiciously leveraged for cargo conjugation to generate the nanoassembly and macrophage targeting, respectively. A responsive linker is engineered into the nanoassembly for releasing the protein cargo inside the macrophages, while ensuring stability during delivery. The design principles are simple and versatile to be applicable to a range of biologics, from small protein toxins to large enzymes, with high loading capacity. This self-assembly platform has the potential for delivering biologics to immune cells with implications in immunotherapy.

Using Live-Cell Imaging to Measure the Effects of Pathological Proteins on Axonal Transport in Primary Hippocampal Neurons.

Bidirectional transport of cargos along the axon is critical for maintaining functional synapses, neural connectivity, and healthy neurons. Axonal transport is disrupted in multiple neurodegenerative diseases, and projection neurons are particularly vulnerable because of the need to transport cellular materials over long distances and sustain substantial axonal mass. Pathological modifications of several disease-related proteins negatively affect transport, including tau, amyloid-β, α-synuclein, superoxide dismutase, and huntingtin, providing a potential common mechanism by which pathological proteins exert toxicity in disease. Methods to study these toxic mechanisms are necessary to understand neurodegenerative disorders and identify potential therapeutic interventions. Here, cultured primary rodent hippocampal neurons are co-transfected with multiple plasmids to study the effects of pathological proteins on fast axonal transport using live-cell confocal imaging of fluorescently-tagged cargo proteins. We begin with the harvest, dissociation, and culturing of primary hippocampal neurons from rodents. Then, we co-transfect the neurons with plasmid DNA constructs to express fluorescent-tagged cargo protein and wild-type or mutant tau (used as an exemplar of pathological proteins). Axons are identified in live cells using an antibody that binds an extracellulardomain of neurofascin, an axon initial segment protein, and an axonal region of interest is imaged to measure fluorescent cargo transport. Using KymoAnalyzer, a freely available ImageJ macro, we extensively characterize the velocity, pause frequency, and directional cargo density of axonal transport, all of which may be affected by the presence of pathological proteins. Through this method, we identify a phenotype of increased cargo pause frequency associated with the expression of pathological tau protein. Additionally, gene-silencing shRNA constructs can be added to the transfection mix to test the role of other proteins in mediating transport disruption. This protocol is easily adaptable for use with other neurodegenerative disease-related proteins and is a reproducible method to study the mechanisms of how those proteins disrupt axonal transport.

Effect of miR-21 in mesenchymal stem cells-derived extracellular vesicles behavior

BackgroundA challenging new branch of research related to aging-associated diseases is the identification of miRNAs capable of modulating the senescence-associated secretory phenotype (SASP) which characterizes senescent cells and contributes to driving inflammation.MethodsMesenchymal stem cells (MSC) from human umbilical cord stroma were stable modified using lentivirus transduction to inhibit miR-21-5p and shotgun proteomic analysis was performed in the MSC-derived extracellular vesicles (EV) to check the effect of miR-21 inhibition in their protein cargo. Besides, we studied the paracrine effect of those modified extracellular vesicles and also their effect on SASP.ResultsSyndecan-1 (SDC1) was the most decreased protein in MSC-miR21−-derived EV, and it was involved in inflammation and EV production. MSC-miR21−-derived EV were found to produce a statistically significant inhibitory effect on SASP and inflammaging markers expression in receptor cells, and in the opposite way, these receptor cells increased their SASP and inflammaging expression statistically significantly when treated with MSC-miR-21+-derived EV.ConclusionThis work demonstrates the importance of miR-21 in inflammaging and its role in SASP through SDC1.Graphical abstract

Targeting ER Acetylation to Rescue Proteotoxicity

Transient acetylation within the endoplasmic reticulum (ER) lumen has emerged as a novel mechanism that regulates proteostasis and dynamics of the secretory pathway. The ER acetylation machinery includes AT-1, a membrane transporter that imports acetyl-CoA from the cytosol into the ER lumen, and two acetyltrasferases, ATase1 and ATase2, which acetylate ER cargo and resident proteins. Loss-of-function mutations or gene duplication events that affect ER acetylation are linked to developmental delay and premature death (homozygous mutation), hereditary forms of peripheral neuropathy (homozygous mutation), and autism spectrum disorder with intellectual disability and progeria (gene duplication). Mouse models of dysfunctional ER acetylation mimic associated human phenotypes.

Proteomic profiling of serum exosomes reveals acute phase response and promotion of inflammatory and platelet activation pathways in patients with heat stroke.

Background: The pathological mechanism of heat stroke (HS) involves the acute phase response, unbalanced immunological/inflammatory reactions, and coagulation initiation, especially platelet activation. Although exosomes contain proteins involved in these biological processes, their protein cargo levels and potential roles in HS remain unknown. This study explored the serum exosome protein expression patterns after HS and their potential roles in the pathogenesis of HS. Methods: Blood samples were collected from ten patients diagnosed with HS upon admission to the intensive care unit (six with severe HS and four with mild HS). Samples from six healthy volunteers were included as control. Using ultracentrifugation, exosomes were prudently isolated, and their protein contents were profiled using liquid chromatography-tandem mass spectrometry analysis with isobaric tags for relative and absolute quantification-based proteomics. Results: Compared with healthy volunteers, patients with HS showed significant changes in the levels of 33 exosomal proteins (23 upregulated and 10 downregulated). The most upregulated proteins included serum amyloid A-1 (SAA-1), von Willebrand factor (vWF), S100A8, and histone H3. In addition, SAA-1, vWF, platelet membrane glycoprotein, S100A8, and histone H3 were more enriched in the exosomes from patients with severe HS than from those with mild HS. Gene ontology analysis revealed that the HS-modulated exosomal proteins were mostly related to inflammatory response, including the acute-phase response, platelet activation/degranulation, and innate immune response. Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed significant enrichment of proteins in the IL-17 signaling pathway, platelet activation, neutrophil extracellular trap formation, Fc epsilon RI signaling pathway, chemokine signaling pathway, and NOD-like receptor signaling pathway, among others. Several serum exosomal proteins, including SAA-1, vWF, and S100A8, which are related to the acute phase, inflammatory response, and platelet activation, were confirmed to be elevated in patients with HS, and were significantly correlated with disease severity, organ dysfunction, and death. Conclusion: Overall, this study explores the potential role of the serum exosomal proteome in the inflammatory response and platelet activation in HS, suggests the pathological mechanisms underlying HS-induced injuries, and recommends reliable exosomal biomarkers for predicting HS prognosis.

Plant Extracellular Vesicles: Current Landscape and Future Directions.

Plant cells secrete membrane-enclosed micrometer- and nanometer-sized vesicles that, similarly to the extracellular vesicles (EVs) released by mammalian or bacterial cells, carry a complex molecular cargo of proteins, nucleic acids, lipids, and primary and secondary metabolites. While it is technically complicated to isolate EVs from whole plants or their tissues, in vitro plant cell cultures provide excellent model systems for their study. Plant EVs have been isolated from the conditioned culture media of plant cell, pollen, hairy root, and protoplast cultures, and recent studies have gathered important structural and biological data that provide a framework to decipher their physiological roles and unveil previously unacknowledged links to their diverse biological functions. The primary function of plant EVs seems to be in the secretion that underlies cell growth and morphogenesis, cell wall composition, and cell-cell communication processes. Besides their physiological functions, plant EVs may participate in defence mechanisms against different plant pathogens, including fungi, viruses, and bacteria. Whereas edible and medicinal-plant-derived nanovesicles isolated from homogenised plant materials ex vivo are widely studied and exploited, today, plant EV research is still in its infancy. This review, for the first time, highlights the different in vitro sources that have been used to isolate plant EVs, together with the structural and biological studies that investigate the molecular cargo, and pinpoints the possible role of plant EVs as mediators in plant-pathogen interactions, which may contribute to opening up new scenarios for agricultural applications, biotechnology, and innovative strategies for plant disease management.

Interrogation and validation of the interactome of neuronal Munc18-interacting Mint proteins with AlphaFold2

Munc18-interacting proteins (Mints) are multidomain adaptors that regulate neuronal membrane trafficking, signaling, and neurotransmission. Mint1 and Mint2 are highly expressed in the brain with overlapping roles in the regulation of synaptic vesicle fusion required for neurotransmitter release by interacting with the essential synaptic protein Munc18-1. Here, we have used AlphaFold2 to identify and then validate the mechanisms that underpin both the specific interactions of neuronal Mint proteins with Munc18-1 as well as their wider interactome. We found that a short acidic α-helical motif within Mint1 and Mint2 is necessary and sufficient for specific binding to Munc18-1 and binds a conserved surface on Munc18-1 domain3b. In Munc18-1/2 double knockout neurosecretory cells, mutation of the Mint-binding site reduces the ability of Munc18-1 to rescue exocytosis, and although Munc18-1 can interact with Mint and Sx1a (Syntaxin1a) proteins simultaneously invitro, we find that they have mutually reduced affinities, suggesting an allosteric coupling between the proteins. Using AlphaFold2 to then examine the entire cellular network of putative Mint interactors provides a structural model for their assembly with a variety of known and novel regulatory and cargo proteins including ADP-ribosylation factor (ARF3/ARF4) small GTPases and the AP3 clathrin adaptor complex. Validation of Mint1 interaction with a new predicted binder TJAP1 (tight junction-associated protein 1) provides experimental support that AlphaFold2 can correctly predict interactions across such large-scale datasets. Overall, our data provide insights into the diversity of interactions mediated by the Mint family and show that Mints may help facilitate a key trigger point in SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor) complex assembly and vesicle fusion.

Structural basis of nuclear transport for NEIL DNA glycosylases mediated by importin-alpha

NEIL glycosylases, including NEIL1, NEIL2, and NEIL3, play a crucial role in the base excision DNA repair pathway (BER). The classical importin pathway mediated by importin α/β and cargo proteins containing nuclear localization sequences (NLS) is the most common transport mechanism of DNA repair proteins to the nucleus. Previous studies have identified putative NLSs located at the C-terminus of NEIL3 and NEIL1. Crystallographic, bioinformatics, calorimetric (ITC), and fluorescence assays were used to investigate the interaction between NEIL1 and NEIL3 putative NLSs and importin-α (Impα). Our findings showed that NEIL3 contains a typical cNLS, with medium affinity for the major binding site of Impα. In contrast, crystallographic analysis of NEIL1 NLS revealed its binding to Impα, but with high B-factors and a lack of electron density at the linker region. ITC and fluorescence assays indicated no detectable affinity between NEIL1 NLS and Impα. These data suggest that NEIL1 NLS is a non-classical NLS with low affinity to Impα. Additionally, we compared the binding mode of NEIL3 and NEIL1 with Mus musculus Impα to human isoforms HsImpα1 and HsImpα3, which revealed interesting binding differences for HsImpα3 variant. NEIL3 is a classical medium affinity monopartite NLS, while NEIL1 is likely to be an unclassical low-affinity bipartite NLS. The base excision repair pathway is one of the primary systems involved in repairing DNA. Thus, understanding the mechanisms of nuclear transport of NEIL proteins is crucial for comprehending the role of these proteins in DNA repair and disease development.

Proteomic profiling of membrane vesicles from Mycobacterium avium subsp. paratuberculosis: Navigating towards an insilico design of a multi-epitope vaccine targeting membrane vesicle proteins

Bacteria typically produce membrane vesicles (MVs) at varying levels depending on the surrounding environments. Gram-negative bacterial outer membrane vesicles (OMVs) have been extensively studied for over 30 years, but MVs from Gram-positive bacteria only recently have been a focus of research. In the present study, we isolated MVs from Mycobacterium avium subsp. paratuberculosis (MAP) and analyzed their protein composition using LC-MS/MS. A total of 316 overlapping proteins from two independent preparations were identified in our study, and topology prediction showed these cargo proteins have different subcellular localization patterns. When MVs were administered to bovine-derived macrophages, significant up-regulation of pro-inflammatory cytokines was observed via qRT-PCR. Proteome functional annotation revealed that many of these proteins are involved in the cellular protein metabolic process, tRNA aminoacylation, and ATP synthesis. Secretory proteins with high antigenicity and adhesion capability were mapped for B-cell and T-cell epitopes. Antigenic, Immunogenic and IFN-γ inducing B-cell, MHC-I, and MHC-II epitopes were stitched together through linkers to form multi-epitope vaccine (MEV) construct against MAP. Strong binding energy was observed during the docking of the 3D structure of the MEV with the bovine TLR2, suggesting that the putative MEV may be a promising vaccine candidate against MAP. However, in vitro and in vivo analysis is required to prove the immunogenic concept of the MEV which we will follow in our future studies. SignificanceJohne's disease is a chronic infection caused by Mycobacterium avium subsp. paratuberculosis that has a potential link to Crohn's disease in humans. The disease is characterized by persistent diarrhea and enteritis, resulting in significant economic losses due to reduced milk yield and premature culling of infected animals. The dairy industry in the United States alone experiences losses of approximately USD 250 million due to Johne's disease. The current vaccine against Johne's disease is limited by several factors, including variable efficacy, limited duration of protection, interference with diagnostic tests, inability to prevent infection, and logistical and cost-related challenges. Nevertheless, a multiepitope vaccine design approach targeting M. avium subsp. paratuberculosis has the potential to overcome these challenges and offer improved protection against Johne's disease.

Effects of Ureaplasma parvum infection in the exosome biogenesis-related proteins in ectocervical epithelial cells.

Ureaplasma parvum is a mycoplasma commonly associated with female reproductive pathologies, such as preterm birth and infertility. It can survive intracellularly and utilize exosomes to propagate infection and its virulence factors. This study explored the differential protein composition of exosomes derived from normal and U. parvum-infected cells. We also investigated the impact of U. parvum on exosome biogenesis in ectocervical epithelial cells. Ectocervical epithelial (ECTO) cells were infected with U. parvum, and immunocytochemical staining was performed using U. parvum-specific marker multiple banded antigen (mba) and exosome marker CD9. NanoLC-MS/MS analysis was conducted to identify differentially expressed proteins in exosomes. Ingenuity Pathway Analysis (IPA) was performed to identify affected canonical pathways and biological functions associated with the protein cargo of exosomes. Western blot analysis of ECTO cells validated the proteomic findings in ECTO cells. U. parvum exhibited colonization of ECTO cells and colocalization with CD9-positive intraluminal vesicles. Proteomic analysis revealed decreased protein abundance and distinct protein profiles in exosomes derived from U. parvum-infected ECTO cells. Differentially expressed proteins were associated with clathrin-mediated endocytosis and various signaling pathways indicative of infection, inflammation, and cell death processes. Additionally, U. parvum infection altered proteins involved in exosome biogenesis. In ECTO cells, U. parvum infection significantly decreased clathrin, ALIX, CD9, and CD63 and significantly increased TSG101, Rab5, Rab35, and UGCG. These findings contribute to our understanding of the infection mechanism and shed light on the importance of exosome-mediated communication in the pathophysiology of diseases affecting the cervix, such as cervicitis and preterm birth.