Cell signaling governs the basic functions of cells by molecular interactions that involve of many proteins. The abundance of signaling proteins can directly influence cellular responses to external ...signal, contributing to cellular heterogeneity. Absolute quantification of proteins is important for modeling and understanding the complex signaling network. Here, we introduce how to measure the amount of TGF-β signaling proteins using quantitative immunoblotting. In addition, we discuss how to convert the measurements of protein abundance to the quantities of absolute molecules per cell. This method is generally applicable to the absolute quantification of other proteins.
Antibody use is a critical component of cardiovascular physiology research, and antibodies are used to monitor protein abundance (immunoblot analysis) and protein expression and localization (in ...tissue by immunohistochemistry and in cells by immunocytochemistry). With ongoing discussions on how to improve reproducibility and rigor, the goal of this review is to provide best practice guidelines regarding how to optimize antibody use for increased rigor and reproducibility in both immunoblot analysis and immunohistochemistry approaches. Listen to this article's corresponding podcast at http://ajpheart.podbean.com/e/guidelines-on-antibody-use-in-physiology-studies/ .
Abstract
Stress granules (SGs) are dynamic, membrane-less organelles. With their formation and disassembly processes characterized, it remains elusive how compositional transitions are coordinated ...during prolonged stress to meet changing functional needs. Here, using time-resolved proteomic profiling of the acute to prolonged heat-shock SG life cycle, we identify dynamic SG proteins, further segregated into early and late proteins. Comparison of different groups of SG proteins suggests that their biochemical properties help coordinate SG compositional and functional transitions. In particular, early proteins, with high phase-separation-propensity, drive the rapid formation of the initial SG platform, while late proteins are subsequently recruited as discrete modules to further functionalize SGs. This model, supported by immunoblotting and immunofluorescence imaging, provides a conceptual framework for the compositional transitions throughout the acute to prolonged SG life cycle. Additionally, an early SG constituent, non-muscle myosin II, is shown to promote SG formation by increasing SG fusion, underscoring the strength of this dataset in revealing the complexity of SG regulation.
Adult-onset inflammatory syndromes often manifest with overlapping clinical features. Variants in ubiquitin-related genes, previously implicated in autoinflammatory disease, may define new disorders.
...We analyzed peripheral-blood exome sequence data independent of clinical phenotype and inheritance pattern to identify deleterious mutations in ubiquitin-related genes. Sanger sequencing, immunoblotting, immunohistochemical testing, flow cytometry, and transcriptome and cytokine profiling were performed. CRISPR-Cas9-edited zebrafish were used as an in vivo model to assess gene function.
We identified 25 men with somatic mutations affecting methionine-41 (p.Met41) in UBA1, the major E1 enzyme that initiates ubiquitylation. (The gene
lies on the X chromosome.) In such patients, an often fatal, treatment-refractory inflammatory syndrome develops in late adulthood, with fevers, cytopenias, characteristic vacuoles in myeloid and erythroid precursor cells, dysplastic bone marrow, neutrophilic cutaneous and pulmonary inflammation, chondritis, and vasculitis. Most of these 25 patients met clinical criteria for an inflammatory syndrome (relapsing polychondritis, Sweet's syndrome, polyarteritis nodosa, or giant-cell arteritis) or a hematologic condition (myelodysplastic syndrome or multiple myeloma) or both. Mutations were found in more than half the hematopoietic stem cells, including peripheral-blood myeloid cells but not lymphocytes or fibroblasts. Mutations affecting p.Met41 resulted in loss of the canonical cytoplasmic isoform of UBA1 and in expression of a novel, catalytically impaired isoform initiated at p.Met67. Mutant peripheral-blood cells showed decreased ubiquitylation and activated innate immune pathways. Knockout of the cytoplasmic UBA1 isoform homologue in zebrafish caused systemic inflammation.
Using a genotype-driven approach, we identified a disorder that connects seemingly unrelated adult-onset inflammatory syndromes. We named this disorder the VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome. (Funded by the NIH Intramural Research Programs and the EU Horizon 2020 Research and Innovation Program.).
Immunoblotting is a ubiquitous immunological technique that aids in detecting and quantifying proteins (including those of lower abundance) and their posttranslational modifications such as ...phosphorylation, acetylation, ubiquitylation, and sumoylation. The technique involves electrophoretically separating proteins on an SDS-PAGE gel, transferring them onto a PVDF (or nitrocellulose) membrane and probing with specific antibodies. Here we describe an immunoblotting technique for detecting cellular phosphohistidine, a labile posttranslational modification, by optimizing experimental conditions such that the labile phosphohistidine signal is conserved throughout the experiment.
Rheumatoid arthritis (RA) is an autoimmune inflammatory disease. Early diagnosis can prevent joint erosion. However, available biomarkers do not always allow for clear distinction between RA and ...non‐RA individuals. It has become known that bacteria/viruses are among the environmental triggers that initiate RA via multiple molecular mechanisms.
Thus, to better understand the role of bacteria in RA, we synthetized 6 peptidomimetics of bacterial ureases' flap region. These peptides were then used to distinguish RA patients from healthy people sera by immunoblotting. Most patients' sera were bound to peptidomimetic characteristic for Enterobacter sp. and Klebsiella sp. flap urease. We also found similarities between peptidomimetic sequence and human proteins connected with RA. This pilot study suggests that bacteria may trigger RA via mechanism of molecular mimicry of urease to host proteins and ureases flap peptidomimetics may be potential candidate as a new additional diagnostic test.
Synthetic peptides mimicking flap region of bacterial ureases are recognized by antibodies in human sera. Significantly higher level of those antibodies is present in RA patients. Peptidomimetics characteristic for Enterobacter sp. and Klebsiella sp. are the most utile in sera differentiation and may have potential as diagnostic test.
We aimed to compare various methods for free light chain (fLC) quantitation in cerebrospinal fluid (CSF) and serum and to determine whether quantitative CSF measurements could reliably predict ...intrathecal fLC synthesis. In addition, we wished to determine the relationship between free kappa and free lambda light chain concentrations in CSF and serum in various disease groups.
We analysed 166 paired CSF and serum samples by at least one of the following methods: turbidimetry (Freelite™, SPAPLUS), nephelometry (N Latex FLC™, BN ProSpec), and two different (commercially available and in-house developed) sandwich ELISAs. The results were compared with oligoclonal fLC detected by affinity-mediated immunoblotting after isoelectric focusing.
Although the correlations between quantitative methods were good, both proportional and systematic differences were discerned. However, no major differences were observed in the prediction of positive oligoclonal fLC test. Surprisingly, CSF free kappa/free lambda light chain ratios were lower than those in serum in about 75% of samples with negative oligoclonal fLC test. In about a half of patients with multiple sclerosis and clinically isolated syndrome, profoundly increased free kappa/free lambda light chain ratios were found in the CSF.
Our results show that using appropriate method-specific cut-offs, different methods of CSF fLC quantitation can be used for the prediction of intrathecal fLC synthesis. The reason for unexpectedly low free kappa/free lambda light chain ratios in normal CSFs remains to be elucidated. Whereas CSF free kappa light chain concentration is increased in most patients with multiple sclerosis and clinically isolated syndrome, CSF free lambda light chain values show large interindividual variability in these patients and should be investigated further for possible immunopathological and prognostic significance.
Given the wide adoption of polydimethylsiloxane (PDMS) for the rapid fabrication of microfluidic networks and the utility of polyacrylamide gel electrophoresis (PAGE), we develop a technique for ...fabrication of PAGE molecular sieving gels in PDMS microchannel networks. In developing the fabrication protocol, we trade-off constraints on materials properties of these two polymer materials: PDMS is permeable to O2 and the presence of O2 inhibits the polymerization of polyacrylamide. We present a fabrication method compatible with performing PAGE protein separations in a composite PDMS-glass microdevice, that toggles from an “enclosed” microchannel for PAGE and blotting to an “open” PA gel lane for immunoprobing and readout. To overcome the inhibitory effects of O2, we coat the PDMS channel with a 10% benzophenone solution, which quenches the inhibiting effect of O2 when exposed to UV, resulting in a PAGE-in-PDMS device. We then characterize the PAGE separation performance. Using a ladder of small-to-mid mass proteins (Trypsin Inhibitor (TI); Ovalbumin (OVA); Bovine Serum Albumin (BSA)), we observe resolution of the markers in <60 s, with separation resolution exceeding 1.0 and CVs of 8.4% for BSA-OVA and 2.4% for OVA-TI, with comparable reproducibility to glass microdevice PAGE. We show that benzophenone groups incorporated into the gel through methacrylamide can be UV-activated multiple times to photocapture protein. PDMS microchannel network is reversibly bonded to a glass slide allowing direct access to separated proteins and subsequent in situ diffusion-driven immunoprobing and total protein Sypro red staining. We see this PAGE-in-PDMS fabrication technique as expanding the application and use of microfluidic PAGE without the need for a glass microfabrication infrastructure.
We report the identification and characterization of a five-carbon protein posttranslational modification (PTM) called lysine glutarylation (Kglu). This protein modification was detected by ...immunoblot and mass spectrometry (MS), and then comprehensively validated by chemical and biochemical methods. We demonstrated that the previously annotated deacetylase, sirtuin 5 (SIRT5), is a lysine deglutarylase. Proteome-wide analysis identified 683 Kglu sites in 191 proteins and showed that Kglu is highly enriched on metabolic enzymes and mitochondrial proteins. We validated carbamoyl phosphate synthase 1 (CPS1), the rate-limiting enzyme in urea cycle, as a glutarylated protein and demonstrated that CPS1 is targeted by SIRT5 for deglutarylation. We further showed that glutarylation suppresses CPS1 enzymatic activity in cell lines, mice, and a model of glutaric acidemia type I disease, the last of which has elevated glutaric acid and glutaryl-CoA. This study expands the landscape of lysine acyl modifications and increases our understanding of the deacylase SIRT5.
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•Lysine glutarylation is a protein posttranslational modification•SIRT5 can catalyze the enzymatic removal of lysine glutarylation•Proteomic analyses identify a link between lysine glutarylation and metabolism•Glutarylation suppresses CPS1 activity, which is targeted by SIRT5 for removal
Tan et al. report a new type of evolutionarily conserved posttranslational modification, lysine glutarylation, targeted by SIRT5 which impacts metabolic processes. CPS1, the rate-limiting enzyme in urea cycle, is suppressed by glutarylation in glutaric academia type I disease and is targeted by SIRT5 for deglutarylation.
Missense mutations in glucocerebrosidase (GBA1) that impair the activity of the encoded lysosomal lipid metabolism enzyme (GCase) are linked to an increased risk of Parkinson's disease. However, ...reduced GCase activity is also found in brain tissue from Parkinson's disease patients without GBA1 mutations, implicating GCase dysfunction in the more common idiopathic form of Parkinson's disease. GCase is very highly expressed in monocytes, and thus we measured GCase activity in blood samples from recently diagnosed Parkinson's disease patients. Flow cytometry and immunoblotting assays were used to measure levels of GCase activity and protein in monocytes and lymphocytes from patients with Parkinson's disease (n = 48) and matched controls (n = 44). Gene sequencing was performed to screen participants for GBA1 missense mutations. In the Parkinson's disease patients, GCase activity was significantly reduced in monocytes, but not lymphocytes, compared to controls, even when GBA1 mutation carriers were excluded. Monocyte GCase activity correlated with plasma ceramide levels in the Parkinson's disease patients. Our results add to evidence for GCase dysfunction in idiopathic Parkinson's disease and warrant further work to determine if monocyte GCase activity associates with Parkinson's disease progression.
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