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Extracellular vesicles, including exosomes, are released by all cells, including those of the nervous system. Capable of delivering lipid, protein and nucleic acids to both nearby and distal cells, exosomes have been hypothesized to play a role in progression of many diseases of the nervous system. To date, most analyses on the role of these vesicles in the healthy and diseased state have relied on studying vesicles from in vitro sources, such as conditioned cell culture media, or body fluids. Here we have taken a critical approach to the enrichment and characterization of exosomes from human frontal cortex. This method maintains the integrity of the vesicles and their cargo, and comprehensive proteomic and genomic characterization confirms the legitimacy of the resulting extracellular vesicles as endosome-derived exosomes. This method will enable neuroscientists to acquire more detailed information about exosomes in the brain and explore the role(s) this form of intercellular communication and unique source of lipid, protein and RNA has in healthy brain function and pathogenic conditions. Furthermore, this method may have important utility in the isolation of exosomes from other tissues.

Exosomes are extracellular vesicles secreted by multiple cell types into the extracellular space. They contain cell-state specific cargos which often reflects the (patho)physiological condition of the cells/organism. Milk contains high amounts of exosomes and it is unclear whether their cargo is altered based on the lactation stage of the organism. Here, we isolated exosomes from bovine milk that were obtained at various stages of lactation and examined the content by quantitative proteomics. Exosomes were isolated by OptiPrep density gradient centrifugation from milk obtained from cow after 24, 48 and 72 h post calving. As control, exosomes were also isolated from cows during mid-lactation period which has been referred to as mature milk (MM). Biochemical and biophysical characterization of exosomes revealed the high abundance of exosomes in colostrum and MM samples. Quantitative proteomics analysis highlighted the change in the proteomic cargo of exosomes based on the lactation state of the cow. Functional enrichment analysis revealed that exosomes from colostrum are significantly enriched with proteins that can potentially regulate the immune response and growth. This study highlights the importance of exosomes in colostrum and hence opens up new avenues to exploit these vesicles in the regulation of the immune response and growth.

Methods that assay protein foldedness with proteomics have generated censuses of apparent protein folding stabilities in biological milieu. However, different censuses poorly correlate with each other. Here, we show that the reason for this is that methods targeting foldedness through monitoring amino acid sidechain reactivity also detect changes in conformation and ligand binding, which can be a substantial fraction of the data. We show that the reactivity of only one quarter of cysteine or methionine sidechains in proteins in a urea denaturation curve of mammalian cell lysate can be confidently explained by a two-state unfolding isotherm. Contrary to that expected from unfolding, up to one third of the cysteines decreased reactivity. These cysteines were enriched in proteins with functions relating to unfolded protein stress. One protein, chaperone HSPA8, displayed changes arising from ligand and cofactor binding. Unmasking this hidden information using the approaches outlined here should improve efforts to understand both folding and the remodeling of protein function directly in complex biological settings. Proteomics can define features of proteome foldedness by assessing the reactivity of surface exposed amino acids. Here, the authors show that such exposure patterns yield insight to structural changes in chaperones as they bind to unfolded proteins in urea-denatured mammalian cell lysate.

Bioinformatics tools are imperative for the in depth analysis of heterogeneous high-throughput data. Most of the software tools are developed by specific laboratories or groups or companies wherein they are designed to perform the required analysis for the group. However, such software tools may fail to capture \"what the community needs in a tool\". Here, we describe a novel community-driven approach to build a comprehensive functional enrichment analysis tool. Using the existing FunRich tool as a template, we invited researchers to request additional features and/or changes. Remarkably, with the enthusiastic participation of the community, we were able to implement 90% of the requested features. FunRich enables plugin for extracellular vesicles wherein users can download and analyse data from Vesiclepedia database. By involving researchers early through community needs software development, we believe that comprehensive analysis tools can be developed in various scientific disciplines.

Dental pulp stem cells (DPSCs) are increasingly recognised for their potential in regenerative medicine, particularly in oral and maxillofacial surgeries. However, the unique microbial environment of the oral cavity necessitates a better understanding of how bacterial challenges may affect DPSC function to optimise their reparative capabilities and potential clinical/therapeutic use. This study focused on identifying and understanding differentially expressed proteins in response to Porphyromonas gingivalis challenge and utilises comparative proteomic methodology of the secretome and whole cell lysates with deeper analysis of differentially expressed proteins using STRINGDB and REACTOME databases to elucidate key proteins and networks. This study identified significant proteomic changes in human DPSCs in response to Pg infection, particularly in growth factor signalling, immune response, and wound healing. These insights advance our understanding of DPSC-pathogen interactions, promoting their potential for regenerative and immunomodulatory therapies against infection.

Pancreatic ductal adenocarcinoma (PDA) exhibits diverse molecular aberrancies that contribute to its aggressive behaviour and poor patient survival. P-21-activated kinase 1 (PAK1) and PAK4 drive the tumorigenesis of PDA. However, their roles in tumour vasculature and the impact on immune response are unclear. This study aims to investigate the effects of PAK1 and PAK4 on tumour vasculature, immune cell infiltration, and the connection between using PAK1-knockout (KO), PAK4 KO, and wild-type (WT) PDA cells in cell-based and mouse experiments. Tumour tissues isolated from a syngeneic mouse model were immuno-stained to determine the changes in tumour vasculature and immune cell infiltration/activation, followed by a proteomic study to assess biological processes involved. PAK1KO or PAK4KO suppressed tumour growth by reducing angiogenesis while enhancing vascular normalisation, enhanced the infiltration/activation of T-cells and dendritic cells associated with upregulation of ICAM-1 and VCAM-1 in the tumour microenvironment, and stimulated vascular immune crosstalk via an ICAM-1-mediated mechanism. This was supported by proteomic profiles indicating the regulation of endothelial cell and leukocyte trans-endothelial migration in PAK1- or PAK4-knockout tumours. In conclusion, PAK1KO or PAK4KO enhanced tumour vascular normalisation while reducing angiogenesis, stimulating immune cell infiltration and activation to suppress tumour growth.

Extracellular vesicles (EVs) represent a system for the coordinated secretion of a variety of molecular cargo including proteins, lipids, nucleic acids, and metabolites. They have an essential role in intercellular communication in multicellular organisms and have more recently been implicated in host-pathogen interactions. Study of the role for EVs in fungal biology has focused on pathogenic yeasts that are major pathogens in humans. In this study we have expanded the investigation of fungal EVs to plant pathogens, specifically the major cotton pathogen f. sp. . EVs isolated from f. sp. culture medium have a morphology and size distribution similar to EVs from yeasts such as and . A unique feature of the EVs from f. sp. is their purple color, which is predicted to arise from a napthoquinone pigment being packaged into the EVs. Proteomic analysis of f. sp. EVs revealed that they are enriched in proteins that function in synthesis of polyketides as well as proteases and proteins that function in basic cellular processes. Infiltration of f. sp. EVs into the leaves of cotton or plants led to a phytotoxic response. These observations lead to the hypothesis that f. sp. EVs are likely to play a crucial role in the infection process.

Legionella pneumophila utilizes a type IVB secretion system (T4SS) to translocate over 300 effector proteins into host cells, hijacking cellular processes, including those within the mitochondrion. Currently, no Legionella effectors have been identified at the mitochondrial outer membrane, a critical interface between the organelle and the rest of the cell. We screened the Legionella effector repertoire for features of mitochondrial tail-anchored (TA) proteins and identified four putative TA effectors. Among them, Lp PIP (Lpg1625) localizes to the mitochondrial outer membrane and interacts with all three isoforms of protein phosphatase 1 (PP1) via an RVxF motif, functioning as a PP1-interacting protein (PIP). Importantly, PP1 remains catalytically active upon interaction with Lp PIP to dephosphorylate mitochondrial outer membrane proteins. Altering the TA signature to direct Lp PIP to the ER induces ER-recruitment of PP1 and dephosphorylation of ER-resident proteins, indicating that Lp PIP controls PP1 localization and not substrate specificity. This study uncovers a novel pathogen-mediated strategy to modulate PP1 and manipulate the host cell phosphoproteome.

The TIM22 complex mediates the import of hydrophobic carrier proteins into the mitochondrial inner membrane. While the TIM22 machinery has been well characterised in yeast, the human complex remains poorly characterised. Here, we identify Tim29 (C19orf52) as a novel, metazoan-specific subunit of the human TIM22 complex. The protein is integrated into the mitochondrial inner membrane with it’s C-terminus exposed to the intermembrane space. Tim29 is required for the stability of the TIM22 complex and functions in the assembly of hTim22. Furthermore, Tim29 contacts the Translocase of the Outer Mitochondrial Membrane, TOM complex, enabling a mechanism for transport of hydrophobic carrier substrates across the aqueous intermembrane space. Identification of Tim29 highlights the significance of analysing mitochondrial import systems across phylogenetic boundaries, which can reveal novel components and mechanisms in higher organisms. Mitochondria are like tiny bean-shaped “power stations” that provide our cells with the vast majority of the energy that they need. These structures, however, are not self-sufficient and instead rely on proteins and chemicals that are imported from elsewhere in the cell. Two layers of membrane enclose the mitochondria, and transporting proteins across the inner and outer membranes requires large molecular machines embedded within the membranes. One such complex, the TIM22 complex, organizes tunnel-like carrier proteins that in turn ferry chemicals across the inner membrane to fuel metabolism. The TIM22 complex is vitally important as it allows mitochondria to adapt their metabolism – that is, how and when they generate energy – to match the cell’s needs during development. Yet, while the TIM22 complex has been studied extensively in yeast, less is known about how it works in human cells. Now, Kang et al. have identified a new piece of the human equivalent of the TIM22 machinery, a protein called Tim29, which helps to assemble the TIM22 complex in human cells. Experiments reveal that Tim29 also creates a link between human TIM22 and the TOM complex, a complex that serves as the general entry point through the outer mitochondrial membrane. Sequence analysis revealed that Tim29 is found in other animals, such as chimpanzees and cows, but not in yeast. This suggests that the mitochondrial machinery has changed during evolution. Kang et al. plan to further investigate how human carrier proteins reach the mitochondria, and exactly how Tim29 helps human TIM22 to cooperate with TOM. Overall, the discovery of Tim29 highlights the importance of looking at mitochondrial machinery across different species in the hope of revealing new components and mechanisms. A future challenge will be to determine how relevant these machines are in human development and diseases.

Background/Objectives: Pancreatic ductal adenocarcinoma (PDA) is one of the most malignant solid cancers. KRAS mutation accounts for over 90% of cases. p21-activated kinases (PAKs) act downstream of KRAS and are involved in tumorigenesis. The inhibition of PAK4 suppresses PDA by stimulating the tumor infiltration of cytotoxic T cells. The major histocompatibility complex class I (MHC I) is a key in presenting antigens to cytotoxic T cells. MHC I degradation via autophagy promotes the immune evasion of pancreatic cancer. We investigated the effect of PAK4 inhibition on MHC I expression and autophagy. Methods: In this study, using proteomic analysis, fluorescence-activated cell sorting (FACS), and immunoblotting, we examined the effect of PAK4 knockout (KO) in human PDA cells on the expression of MHC I and autophagy to identify the mechanism involved in the stimulation of cytotoxic T cells by PAK4 inhibition. Results: We found that PAK4 KO increased MHC I expression in two human PDA cell lines: MiaPaCa-2 and PANC-1. PAK4 KO also increased cancer cell autophagy. However, the inhibition of autophagy by chloroquine (CQ) did not affect the effect of PAK4 KO on apoptosis and cell death. More importantly, the inhibition of autophagy by CQ did not alter the expression of MHC I stimulated by PAK4 KO, indicating that PAK4 KO stimulated MHC I expression via an autophagy-independent pathway. Conclusions: We identified a role of PAK4 in MHC I expression by PDA cells, which is independent of autophagy.