Expression study of Krabbe Disease missense variants - Insights from quantification profiles of residual enzyme activity, secretion and psychosine levels.

Krabbe disease (KD) is an autosomal recessive lysosomal storage disorder caused by loss-of-function mutations in the gene, which encodes for the enzyme galactosylceramidase (GALC). GALC is crucial for myelin metabolism. Functional deficiency of GALC leads to toxic accumulation of psychosine, dysfunction and death of oligodendrocytes, and eventual brain demyelination.

Dominant Circulating Cell-free Mycobacterial Proteins in In-use Machining Fluid and their Antigenicity Potential.

Background: Occupational exposure to industrial Metalworking Fluid (MWF) colonized by (MI) has been associated with immune lung disease hypersensitivity pneumonitis (HP) in machinists. This warrants regular fluid monitoring for early detection of mycobacterial proteins, especially those with antigenic potential.

Objective: To detect and identify dominant MI proteins and antigens directly from the field-drawn in-use MWF using an integrated immunoproteomic-immunoinformatic approach.

Predominantly Orphan Secretome in the Lung Pathogen Revealed by a Multipronged Growth-Phase-Driven Strategy.

The emerging lung pathogen is understudied for its virulence determinants and molecular targets for diagnosis and therapeutics. Here, we report a comprehensive secretome (600 proteins) of this species, which was identified using a multipronged strategy based on genetic/genomic, proteomic, and bioinformatic approaches. In-solution digested bottom-up proteomics from various growth phases identified a total of 517 proteins, while 2D-GE proteomics identified 33 proteins.

Structural and functional fine mapping of cysteines in mammalian glutaredoxin reveal their differential oxidation susceptibility.

Protein-S-glutathionylation is a post-translational modification involving the conjugation of glutathione to protein thiols, which can modulate the activity and structure of key cellular proteins. Glutaredoxins (GLRX) are oxidoreductases that regulate this process by performing deglutathionylation. However, GLRX has five cysteines that are potentially vulnerable to oxidative modification, which is associated with GLRX aggregation and loss of activity.

Mitoquinone mesylate attenuates pathological features of lean and obese allergic asthma in mice.

Obesity is associated with severe, difficult-to-control asthma, and increased airway oxidative stress. Mitochondrial reactive oxygen species (mROS) are an important source of oxidative stress in asthma, leading us to hypothesize that targeting mROS in obese allergic asthma might be an effective treatment. Using a mouse model of house dust mite (HDM)-induced allergic airway disease in mice fed a low- (LFD) or high-fat diet (HFD), and the mitochondrial antioxidant MitoQuinone (MitoQ), we investigated the effects of obesity and ROS on HDM-induced airway inflammation, remodeling, and airway hyperresponsiveness (AHR).

Inhibition of PDIA3 in club cells attenuates osteopontin production and lung fibrosis.

Background: The role of club cells in the pathology of idiopathic pulmonary fibrosis (IPF) is not well understood. Protein disulfide isomerase A3 (PDIA3), an endoplasmic reticulum-based redox chaperone required for the functions of various fibrosis-related proteins; however, the mechanisms of action of PDIA3 in pulmonary fibrosis are not fully elucidated.

Objectives: To examine the role of club cells and PDIA3 in the pathology of pulmonary fibrosis and the therapeutic potential of inhibition of PDIA3 in lung fibrosis.

Ciliary Ca2+ pumps regulate intraciliary Ca2+ from the action potential and may co-localize with ciliary voltage-gated Ca2+ channels.

Calcium ions (Ca2+) entering cilia through the ciliary voltage-gated calcium channels (CaV) during the action potential causes reversal of the ciliary power stroke and backward swimming in Paramecium tetraurelia. How calcium is returned to the resting level is not yet clear. Our focus is on calcium pumps as a possible mechanism.

Glutathionylation chemistry promotes interleukin-1 beta-mediated glycolytic reprogramming and pro-inflammatory signaling in lung epithelial cells.

Glycolysis is a well-known process by which metabolically active cells, such as tumor or immune cells meet their high metabolic demands. Previously, our laboratory has demonstrated that in airway epithelial cells, the pleiotropic cytokine, interleukin-1 beta (IL1B) induces glycolysis and that this contributes to allergic airway inflammation and remodeling. Activation of glycolysis is known to increase NADPH reducing equivalents generated from the pentose phosphate pathway, linking metabolic reprogramming with redox homeostasis.

Coactosin Phosphorylation Controls Entamoeba histolytica Cell Membrane Protrusions and Cell Motility.

Invasion of the colon wall by during amoebic dysentery entails migration of trophozoites through tissue layers that are rich in extracellular matrix. Transcriptional silencing of the surface metalloprotease EhMSP-1 produces hyperadherent less-motile trophozoites that are deficient in forming invadosomes. Reversible protein phosphorylation is often implicated in regulation of cell motility and invadosome formation.

Dysregulation of the glutaredoxin/glutathionylation redox axis in lung diseases.

Glutathione is a major redox buffer, reaching millimolar concentrations within cells and high micromolar concentrations in airways. While glutathione has been traditionally known as an antioxidant defense mechanism that protects the lung tissue from oxidative stress, glutathione more recently has become recognized for its ability to become covalently conjugated to reactive cysteines within proteins, a modification known as glutathionylation (or glutathiolation or protein mixed disulfide). glutathionylation has the potential to change the structure and function of the target protein, owing to its size (the addition of three amino acids) and charge (glutamic acid).

Inclusion of grape marc in dairy cattle rations alters the bovine milk proteome.

Grape marc (GPM) is a viticulture by-product that is rich in secondary compounds, including condensed tannins (CT), and is used as a supplement in livestock feeding practices. The aim of this study was to determine whether feeding GPM to lactating dairy cows would alter the milk proteome through changes in nitrogen (N) partitioning. Ten lactating Holstein cows were fed a total mixed ration (TMR) top-dressed with either 1.

Direct cysteine sulfenylation drives activation of the Src kinase.

The Src kinase controls aspects of cell biology and its activity is regulated by intramolecular structural changes induced by protein interactions and tyrosine phosphorylation. Recent studies indicate that Src is additionally regulated by redox-dependent mechanisms, involving oxidative modification(s) of cysteines within the Src protein, although the nature and molecular-level impact of Src cysteine oxidation are unknown. Using a combination of biochemical and cell-based studies, we establish the critical importance of two Src cysteine residues, Cys-185 and Cys-277, as targets for HO-mediated sulfenylation (Cys-SOH) in redox-dependent kinase activation in response to NADPH oxidase-dependent signaling.

Bacterial Lipoproteins Constitute the TLR2-Stimulating Activity of Serum Amyloid A.

Studies comparing endogenous and recombinant serum amyloid A (SAA) have generated conflicting data on the proinflammatory function of these proteins. In exploring this discrepancy, we found that in contrast to commercially sourced recombinant human SAA1 (hSAA1) proteins produced in , hSAA1 produced from eukaryotic cells did not promote proinflammatory cytokine production from human or mouse cells, induce Th17 differentiation, or stimulate TLR2. Proteomic analysis of -derived hSAA1 revealed the presence of numerous bacterial proteins, with several being reported or probable lipoproteins.

Avian lungs: A novel scaffold for lung bioengineering.

Allogeneic lung transplant is limited both by the shortage of available donor lungs and by the lack of suitable long-term lung assist devices to bridge patients to lung transplantation. Avian lungs have different structure and mechanics resulting in more efficient gas exchange than mammalian lungs. Decellularized avian lungs, recellularized with human lung cells, could therefore provide a powerful novel gas exchange unit for potential use in pulmonary therapeutics.

Mouse serum exosomal proteomic signature in response to asbestos exposure.

Asbestos-induced diseases like fibrosis and mesothelioma are very aggressive, without any treatment options. These diseases are diagnosed only at the terminal stages due to lack of early stage biomarkers. The recent discovery of exosomes as circulating biomarkers led us to look for exosomal biomarkers of asbestos exposure in mouse blood.

Exosomes from asbestos-exposed cells modulate gene expression in mesothelial cells.

Asbestos exposure is a determinate cause of many diseases, such as mesothelioma, fibrosis, and lung cancer, and poses a major human health hazard. At this time, there are no identified biomarkers to demarcate asbestos exposure before the presentation of disease and symptoms, and there is only limited understanding of the underlying biology that governs asbestos-induced disease. In our study, we used exosomes, 30-140 nm extracellular vesicles, to gain insight into these knowledge gaps.

TGF-β1-induced deposition of provisional extracellular matrix by tracheal basal cells promotes epithelial-to-mesenchymal transition in a c-Jun NH-terminal kinase-1-dependent manner.

Epithelial cells have been suggested as potential drivers of lung fibrosis, although the epithelial-dependent pathways that promote fibrogenesis remain unknown. Extracellular matrix is increasingly recognized as an environment that can drive cellular responses in various pulmonary diseases. In this study, we demonstrate that transforming growth factor-β1 (TGF-β1)-stimulated mouse tracheal basal (MTB) cells produce provisional matrix proteins in vitro, which initiate mesenchymal changes in subsequently freshly plated MTB cells via Rho kinase- and c-Jun NH-terminal kinase (JNK1)-dependent processes.

Scaling Proteome-Wide Reactions of Activity-Based Probes.

Unified analysis of complex reactions of an activity-based probe with proteins in a proteome remains an unsolved challenge. We propose a power expression, rate = k[Probe], for scaling the progress of proteome-wide reactions and use the scaling factor (0 ≤ α ≤ 1) as an apparent, partial order with respect to the probe to measure the "enzyme-likeness" for a protein in reaction acceleration. Thus, α reports the intrinsic reactivity of the protein with the probe.

Development and Applications of a Calmodulin-Based Fusion Protein System for the Expression and Purification of WW and Zinc Finger Modules.

Calmodulin from is an α-helical calcium-binding protein that expresses to high levels in . When the N-terminus of a calmodulin variant is bound to Ca, it undergoes a conformational change, exposing hydrophobic pockets. This property can be utilized for purification purposes, as these pockets bind to phenyl sepharose resin with high affinity.

Comparative Decellularization and Recellularization of Wild-Type and Alpha 1,3 Galactosyltransferase Knockout Pig Lungs: A Model for Ex Vivo Xenogeneic Lung Bioengineering and Transplantation.

Background: A novel potential approach for lung transplantation could be to utilize xenogeneic decellularized pig lung scaffolds that are recellularized with human lung cells. However, pig tissues express several immunogenic proteins, notably galactosylated cell surface glycoproteins resulting from alpha 1,3 galactosyltransferase (α-gal) activity, that could conceivably prevent effective use. Use of lungs from α-gal knock out (α-gal KO) pigs presents a potential alternative and thus comparative de- and recellularization of wild-type and α-gal KO pig lungs was assessed.

Comparative proteomics dataset of skimmed milk samples from Holstein and Jersey dairy cattle.

Milk samples were collected from Holstein and Jersey breeds of dairy cattle maintained under the same management practices and environmental conditions over a seven-day period. Milk samples were collected twice daily from six cows of each breed as previously described (Tacoma et al., 2016) [1].

Characterization of the bovine milk proteome in early-lactation Holstein and Jersey breeds of dairy cows.

Unlabelled: Milk is a highly nutritious natural product that provides not only a rich source of amino acids to the consumer but also hundreds of bioactive peptides and proteins known to elicit health-benefitting activities. We investigated the milk protein profile produced by Holstein and Jersey dairy cows maintained under the same diet, management and environmental conditions using proteomic approaches that optimize protein extraction and characterization of the low abundance proteins within the skim milk fraction of bovine milk. In total, 935 low abundance proteins were identified.