Liver and pancreatic-targeted interleukin-22 as a therapeutic for metabolic dysfunction-associated steatohepatitis.

Metabolic dysfunction-associated steatohepatitis (MASH) is the most prevalent cause of liver disease worldwide, with a single approved therapeutic. Previous research has shown that interleukin-22 (IL-22) can suppress β-cell stress, reduce local islet inflammation, restore appropriate insulin production, reverse hyperglycemia, and ameliorate insulin resistance in preclinical models of diabetes. In clinical trials long-acting forms of IL-22 have led to increased proliferation in the skin and intestine, where the IL-22RA1 receptor is highly expressed.

Metaproteomic sample preparation methods bias the recovery of host and microbial proteins according to taxa and cellular compartment.

Faecal proteomics studies have focussed on identification of microbial proteins, however; stool represents a valuable resource to interrogate the host interactions with the microbiota without the need for invasive intestinal biopsies. As the widely used enrichment method (differential centrifugation, DC) enriches for microbial proteins, we compared two other methods for enrichment of host proteins, termed 'host enriched' (HE) and ALL (all proteins). The HE and ALL protocols identified 1.

Intestinal Metaproteomics Reveals Host-Microbiota Interactions in Subjects at Risk for Type 1 Diabetes.

Objective: Dysbiosis of the gut microbiota has been linked to disease pathogenesis in type 1 diabetes, yet the functional consequences to the host of this dysbiosis are unknown. We investigated the functional interactions between the microbiota and the host associated with type 1 diabetes disease risk.

Research Design And Methods: We performed a cross-sectional analysis of stool samples from subjects with recent-onset type 1 diabetes ( = 33), islet autoantibody-positive subjects ( = 17), low-risk autoantibody-negative subjects ( = 29), and healthy subjects ( = 22).

Genetic Variants in ERAP1 and ERAP2 Associated With Immune-Mediated Diseases Influence Protein Expression and the Isoform Profile.

Objective: Endoplasmic reticulum aminopeptidase 1 (ERAP-1) and ERAP-2, encoded on chromosome 5q15, trim endogenous peptides for HLA-mediated presentation to the immune system. Polymorphisms in ERAP1 and/or ERAP2 are strongly associated with several immune-mediated diseases with specific HLA backgrounds, implicating altered peptide handling and presentation as prerequisites for autoreactivity against an arthritogenic peptide. Given the thorough characterization of disease risk-associated polymorphisms that alter ERAP activity, this study aimed instead to interrogate the expression effect of chromosome 5q15 polymorphisms to determine their effect on ERAP isoform and protein expression.

Collagen beta (1-) galactosyltransferase 1 (GLT25D1) is required for the secretion of high molecular weight adiponectin and affects lipid accumulation.

Secretion of high molecular weight (HMW) adiponectin is dependent on post-translational modification (PTM) of conserved lysines in the collagenous domain. The present study aims to characterize the enzymes responsible for the PTM of conserved lysines which leads to HMW adiponectin secretion, and to define its significance in relation to obesity. Collagen beta (1-) galactosyltransferase 1 (GLT25D1) was knocked down in HEK cells modified for the stable expression of adiponectin (adiponectin expressing human embryonic kidney cells, Adipo-HEK) as well as in Simpson Golabi-Behmel-Syndrome (SGBS) adipocytes.

Novel insight into the composition of human single-stranded DNA-binding protein 1 (hSSB1)-containing protein complexes.

Background: Single-stranded DNA-binding proteins are essential cellular components required for the protection, metabolism and processing of single-stranded DNA. Human single-stranded DNA-binding protein 1 (hSSB1) is one such protein, with described roles in genome stability maintenance and transcriptional regulation. As yet, however, the mechanisms through which hSSB1 functions and the binding partners with which it interacts remain poorly understood.

Implementation and evaluation of amyloidosis subtyping by laser-capture microdissection and tandem mass spectrometry.

Background: Correct identification of the amyloidosis-causing protein is crucial for clinical management. Recently the Mayo Clinic reported laser-capture microdissection (LCM) with liquid chromatography-coupled tandem mass spectrometry (MS/MS) as a new diagnostic tool for amyloid diagnosis. Here, we report an independent implementation of this proteomic diagnostics method at the Princess Alexandra Hospital Amyloidosis Centre in Brisbane, Australia.

Reducing the cost of semi-automated in-gel tryptic digestion and GeLC sample preparation for high-throughput proteomics.

Peptide generation by trypsin digestion is typically the first step in mass spectrometry-based proteomics experiments, including 'bottom-up' discovery and targeted proteomics using multiple reaction monitoring. Manual tryptic digest and the subsequent clean-up steps can add variability even before the sample reaches the analytical platform. While specialized filter plates and tips have been designed for automated sample processing, the specialty reagents required may not be accessible or feasible due to their high cost.

Extracellular vesicles secreted by highly metastatic clonal variants of osteosarcoma preferentially localize to the lungs and induce metastatic behaviour in poorly metastatic clones.

Osteosarcoma (OS) is the most common pediatric bone tumor and is associated with the emergence of pulmonary metastasis. Unfortunately, the mechanistic basis for metastasis remains unclear. Tumor-derived extracellular vesicles (EVs) have been shown to play critical roles in cell-to-cell communication and metastatic progression in other cancers, but their role in OS has not been explored.

Molecular Characterization of Caveolin-induced Membrane Curvature.

The generation of caveolae involves insertion of the cholesterol-binding integral membrane protein caveolin-1 (Cav1) into the membrane, however, the precise molecular mechanisms are as yet unknown. We have speculated that insertion of the caveolin scaffolding domain (CSD), a conserved amphipathic region implicated in interactions with signaling proteins, is crucial for caveola formation. We now define the core membrane-juxtaposed region of Cav1 and show that the oligomerization domain and CSD are protected by tight association with the membrane in both mature mammalian caveolae and a model prokaryotic system for caveola biogenesis.

JIP4 is a PLK1 binding protein that regulates p38MAPK activity in G2 phase.

Cell cycle progression from G2 phase into mitosis is regulated by a complex network of mechanisms, all of which finally control the timing of Cyclin B/CDK1 activation. PLK1 regulates a network of events that contribute to regulating G2/M phase progression. Here we have used a proteomics approach to identify proteins that specifically bind to the Polobox domain of PLK1.

Senescent human hepatocytes express a unique secretory phenotype and promote macrophage migration.

Aim: To develop a model of stress-induced senescence to study the hepatocyte senescence associated secretory phenotype (SASP).

Methods: Hydrogen peroxide treatment was used to induce senescence in the human HepG2 hepatocyte cell line. Senescence was confirmed by cytochemical staining for a panel of markers including Ki67, p21, heterochromatin protein 1β, and senescence-associated-β-galactosidase activity.

Total transcriptome, proteome, and allergome of Johnson grass pollen, which is important for allergic rhinitis in subtropical regions.

Background: Genomic data are lacking for many allergen sources. To circumvent this limitation, we implemented a strategy to reveal the repertoire of pollen allergens of a grass with clinical importance in subtropical regions, where an increasing proportion of the world's population resides.

Objective: We sought to identify and immunologically characterize the allergenic components of the Panicoideae Johnson grass pollen (JGP; Sorghum halepense).

Online quantitative proteomics p-value calculator for permutation-based statistical testing of peptide ratios.

The utility of high-throughput quantitative proteomics to identify differentially abundant proteins en-masse relies on suitable and accessible statistical methodology, which remains mostly an unmet need. We present a free web-based tool, called Quantitative Proteomics p-value Calculator (QPPC), designed for accessibility and usability by proteomics scientists and biologists. Being an online tool, there is no requirement for software installation.

A mortise-tenon joint in the transmembrane domain modulates autotransporter assembly into bacterial outer membranes.

Bacterial autotransporters comprise a 12-stranded membrane-embedded β-barrel domain, which must be folded in a process that entraps segments of an N-terminal passenger domain. This first stage of autotransporter folding determines whether subsequent translocation can deliver the N-terminal domain to its functional form on the bacterial cell surface. Here, paired glycine-aromatic 'mortise and tenon' motifs are shown to join neighbouring β-strands in the C-terminal barrel domain, and mutations within these motifs slow the rate and extent of passenger domain translocation to the surface of bacterial cells.

Co-regulation of cell polarization and migration by caveolar proteins PTRF/Cavin-1 and caveolin-1.

Caveolin-1 and caveolae are differentially polarized in migrating cells in various models, and caveolin-1 expression has been shown to quantitatively modulate cell migration. PTRF/cavin-1 is a cytoplasmic protein now established to be also necessary for caveola formation. Here we tested the effect of PTRF expression on cell migration.

High-throughput lectin magnetic bead array-coupled tandem mass spectrometry for glycoprotein biomarker discovery.

Alterations in protein glycosylation occur during development and progression of many diseases, hence glycomics and glycoproteomics have emerged as important tools in glycobiomarker discovery. High-throughput glycan profiling can now be achieved with the recent developments in MS-based techniques. To enable identification and rapid monitoring of glycosylation changes in serum proteins, we developed a semi-automated high-throughput glycoprotein biomarker discovery platform termed lectin magnetic bead array-coupled tandem mass spectrometry (LeMBA-MS) which includes (i) effective single-step serum glycoprotein isolation using a panel of 20 individual lectin-coated magnetic beads in microplate format, (ii) on-bead trypsin digestion, and (iii) nanoLC-MS/MS with lectin exclusion list.

Proteomics in molecular diagnosis: typing of amyloidosis.

Amyloidosis is a group of disorders caused by deposition of misfolded proteins as aggregates in the extracellular tissues of the body, leading to impairment of organ function. Correct identification of the causal amyloid protein is absolutely crucial for clinical management in order to avoid misdiagnosis and inappropriate, potentially harmful treatment, to assess prognosis and to offer genetic counselling if relevant. Current diagnostic methods, including antibody-based amyloid typing, have limited ability to detect the full range of amyloid forming proteins.

Expression of PTRF in PC-3 Cells modulates cholesterol dynamics and the actin cytoskeleton impacting secretion pathways.

Expression of caveolin-1 is up-regulated in prostate cancer metastasis and is associated with aggressive recurrence of the disease. Intriguingly, caveolin-1 is also secreted from prostate cancer cell lines and has been identified in secreted prostasomes. Caveolin-1 is the major structural component of the plasma membrane invaginations called caveolae.

Differential impact of caveolae and caveolin-1 scaffolds on the membrane raft proteome.

Caveolae, a class of cholesterol-rich lipid rafts, are smooth invaginations of the plasma membrane whose formation in nonmuscle cells requires caveolin-1 (Cav1). The recent demonstration that Cav1-associated cavin proteins, in particular PTRF/cavin-1, are also required for caveolae formation supports a functional role for Cav1 independently of caveolae. In tumor cells deficient for Golgi β-1,6N-acetylglucosaminyltransferase V (Mgat5), reduced Cav1 expression is associated not with caveolae but with oligomerized Cav1 domains, or scaffolds, that functionally regulate receptor signaling and raft-dependent endocytosis.

The dominant 55 kDa allergen of the subtropical Bahia grass (Paspalum notatum) pollen is a group 13 pollen allergen, Pas n 13.

Bahia grass, Paspalum notatum, is an important pollen allergen source with a long season of pollination and wide distribution in subtropical and temperate regions. We aimed to characterize the 55 kDa allergen of Bahia grass pollen (BaGP) and ascertain its clinical importance. BaGP extract was separated by 2D-PAGE and immunoblotted with serum IgE of a grass pollen-allergic patient.

Lectin magnetic bead array for biomarker discovery.

Alterations in protein glycosylation play an important role in patho-physiology, and much effort has been devoted to detecting glycoprotein biomarkers. In this manuscript, we describe the development of a novel method for monitoring alterations in protein glycosylation. Lectins are used as individual affinity reagents and coupled to magnetic beads (Dynabeads) in a microplate array format for isolation of glycosylated proteins.

Nucleophosmin and nucleolin regulate K-Ras plasma membrane interactions and MAPK signal transduction.

The spatial organization of Ras proteins into nanoclusters on the inner leaflet of the plasma membrane is essential for high fidelity signaling through the MAPK pathway. Here we identify two selective regulators of K-Ras nanoclustering from a proteomic screen for K-Ras interacting proteins. Nucleophosmin (NPM) and nucleolin are predominantly localized to the nucleolus but also have extranuclear functions.