Antibiotic resistance is a major public health threat, and alternatives to antibiotic therapy are urgently needed. Immunotherapy, particularly the blockade of inhibitory immune checkpoints, is a leading treatment option in cancer and autoimmunity. In this study, we used a murine model of Salmonella Typhimurium infection to investigate whether immune checkpoint blockade could be applied to bacterial infection.
View Article and Find Full Text PDFVascular cell adhesion molecule-1 (VCAM-1) has been well established as a critical contributor to atherosclerosis and consequently as an attractive therapeutic target for anti-atherosclerotic drug candidates. Many publications have demonstrated that disrupting the VCAM-1 function blocks monocyte infiltration into the sub-endothelial space, which effectively prevents macrophage maturation and foam cell transformation necessary for atherosclerotic lesion formation. Currently, most VCAM-1-inhibiting drug candidates in pre-clinical and clinical testing do not directly target VCAM-1 itself but rather down-regulate its expression by inhibiting upstream cytokines and transcriptional regulators.
View Article and Find Full Text PDFMonoclonal antibodies (mAbs) are one of the most successful and versatile protein-based pharmaceutical products used to treat multiple pathological conditions. The remarkable specificity of mAbs and their affinity for biological targets has led to the implementation of mAbs in the therapeutic regime of oncogenic, chronic inflammatory, cardiovascular, and infectious diseases. Thus, the discovery of novel mAbs with defined functional activities is of crucial importance to expand our ability to address current and future clinical challenges.
View Article and Find Full Text PDFBiochim Biophys Acta Mol Cell Res
August 2021
The Endoplasmic Reticulum (ER) is responsible for the folding and post-translational modification of secretory proteins, as well as for triaging misfolded proteins. During folding, there is a complex yet only partially understood interplay between disulfide bond formation, which is an enzyme catalyzed event in the oxidizing environment of the ER, along with other post-translational modifications (PTMs) and chaperone-supported protein folding. Here, we used the glycoprotein torsinA as a model substrate to explore the impact of ER redox homeostasis on PTMs and protein biogenesis.
View Article and Find Full Text PDFCoagulation factor IX (FIX) is a complex post-translationally modified human serum glycoprotein and high-value biopharmaceutical. The quality of recombinant FIX (rFIX), especially complete γ-carboxylation, is critical for rFIX clinical efficacy. Bioreactor operating conditions can impact rFIX production and post-translational modifications (PTMs).
View Article and Find Full Text PDFGermin and germin-like proteins (GLPs) are a broad family of extracellular glycoproteins ubiquitously distributed in plants. Overexpression of Oryza sativa root germin like protein 1 (OsRGLP1) enhances superoxide dismutase (SOD) activity in transgenic plants. Here, we report bioinformatic analysis and heterologous expression of OsRGLP1 to study the role of glycosylation on OsRGLP1 protein stability and activity.
View Article and Find Full Text PDFFront Mol Biosci
October 2020
TorsinA is a AAA+ ATPase involved in the severe neurological disease Early Onset Torsion Dystonia. Despite the impressive progress in the field in the recent years, the structural organization and function of this intriguing molecule is still not clear. One outstanding difference between torsinA and other AAA+ ATPases is that torsinA is a glycoprotein.
View Article and Find Full Text PDFHuman Factor IX is a highly post-translationally modified protein that is an important clotting factor in the blood coagulation cascade. Functional deficiencies in Factor IX result in the bleeding disorder haemophilia B, which is treated with plasma-derived or recombinant Factor IX concentrates. Here, we investigated the post-translational modifications of human serum-derived Factor IX and report previously undescribed O-linked monosaccharide compositions at serine 141 and a novel site of glycosylation.
View Article and Find Full Text PDFProteomic analysis of bioreactor supernatants can inform on cellular metabolic status, viability, and productivity, as well as product quality, which can in turn help optimize bioreactor operation. Incubating mammalian cells in bioreactors requires the addition of polymeric surfactants such as Pluronic F68, which reduce the sheer stress caused by agitation. However, these surfactants are incompatible with mass spectrometry proteomics and must be eliminated during sample preparation.
View Article and Find Full Text PDFData Independent Acquisition (DIA) Mass Spectrometry (MS) workflows allow unbiased measurement of all detectable peptides from complex proteomes, but require ion libraries for interrogation of peptides of interest. These DIA ion libraries can be theoretical or built from peptide identification data from Data Dependent Acquisition (DDA) MS workflows. However, DDA libraries derived from empirical data rely on confident peptide identification, which can be challenging for peptides carrying complex post-translational modifications.
View Article and Find Full Text PDFPolysialylation is the enzymatic addition of a highly negatively charged sialic acid polymer to the non-reducing termini of glycans. Polysialylation plays an important role in development, and is involved in neurological diseases, neural tissue regeneration, and cancer. Polysialic acid (PSA) is also a biodegradable and non-immunogenic conjugate to therapeutic drugs to improve their pharmacokinetics.
View Article and Find Full Text PDFGlycosylation is a complex posttranslational modification that is critical for regulating the functions of diverse proteins. Analysis of protein glycosylation is made challenging by the high degree of heterogeneity in both glycan occupancy and structure. Here, we describe methods for data-independent acquisition (SWATH) mass spectrometry analysis of structure and occupancy of N-glycans from yeast cell wall glycoproteins.
View Article and Find Full Text PDFNearly one third of the eukaryotic proteome traverses the secretory pathway and most of these proteins are N-glycosylated in the lumen of the endoplasmic reticulum. N-glycans fulfill multiple structural and biological functions, and are crucial for productive folding of many glycoproteins. N-glycosylation involves the attachment of an oligosaccharide to selected asparagine residues in the sequence N-X-S/T (X ≠ P), a motif known as an N-glycosylation'sequon'.
View Article and Find Full Text PDFDystonia is the third most common movement disorder, but its diagnosis and treatment remain challenging. One of the most severe types of dystonia is early-onset torsion dystonia (EOTD). The best studied and validated EOTD-associated mutation, torsinAΔE, is a deletion of a C-terminal glutamate residue in the AAA+ ATPase torsinA.
View Article and Find Full Text PDFMol Cell Proteomics
July 2016
Glycan macro- and microheterogeneity have profound impacts on protein folding and function. This heterogeneity can be regulated by physiological or environmental factors. However, unregulated heterogeneity can lead to disease, and mutations in the glycosylation process cause a growing number of Congenital Disorders of Glycosylation.
View Article and Find Full Text PDFGlycosylation is a co- and post-translational modification that is critical for the regulation of the biophysical properties and biological activities of diverse proteins. Biosynthetic pathways for protein glycosylation are inherently inefficient, resulting in high structural diversity in mature glycoproteins. Macroheterogeneity is the structural diversity due to the presence or absence of glycans at specific glycosylation sites, and is caused by inefficiency in the initial transfer of glycans to proteins.
View Article and Find Full Text PDFA single GAG codon deletion in the gene encoding torsinA is linked to most cases of early-onset torsion dystonia. TorsinA is an ER-localized membrane-associated ATPase from the AAA+ superfamily with an unknown biological function. We investigated the formation of oligomeric complexes of torsinA in cultured mammalian cells and found that wild type torsinA associates into a complex with a molecular weight consistent with that of a homohexamer.
View Article and Find Full Text PDFEarly-onset torsion dystonia (EOTD) is a neurological disorder characterized by involuntary and sustained muscle contractions that can lead to paralysis and abnormal posture. EOTD is associated with the deletion of a glutamate (ΔE) in torsinA, an endoplasmic reticulum (ER) resident AAA(+) ATPase. To date, the effect of ΔE on torsinA and the reason that this mutation results in EOTD are unclear.
View Article and Find Full Text PDFCandida albicans is the most prevalent fungal pathogen of humans. The current techniques used to construct C. albicans strains require integration of exogenous DNA at ectopic locations, which can exert position effects on gene expression that can confound the interpretation of data from critical experiments such as virulence assays.
View Article and Find Full Text PDFEpigenetic mechanisms regulate the expression of virulence traits in diverse pathogens, including protozoan and fungi. In the human fungal pathogen Candida albicans, virulence traits such as antifungal resistance, white-opaque switching, and adhesion to lung cells are regulated by histone deacetylases (HDACs). However, the role of HDACs in the regulation of the yeast-hyphal morphogenetic transitions, a critical virulence attribute of C.
View Article and Find Full Text PDFChromatin function depends on adequate histone stoichiometry. Alterations in histone dosage affect transcription and chromosome segregation, leading to growth defects and aneuploidies. In the fungal pathogen Candida albicans, aneuploidy formation is associated with antifungal resistance and pathogenesis.
View Article and Find Full Text PDFThe success of Candida albicans as a major human fungal pathogen is dependent on its ability to colonize and survive as a commensal on diverse mucosal surfaces. One trait required for survival and virulence in the host is the morphogenetic yeast-to-hypha transition. Mds3 was identified as a regulator of pH-dependent morphogenesis that functions in parallel with the classic Rim101 pH-sensing pathway.
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