Proteins are large, complex molecules that regulate multiple functions within the cell. The protein rarely functions as a single molecule, but rather interacts with one or more other proteins forming ...a dynamic network. Protein-protein interactions are critical for regulating the cell's response toward various stimuli from outside and inside the cell. Identification of protein-protein interactions enhanced our understanding of various biological processes in the living cell. Immunoprecipitation (IP) has been one of the standard and most commonly used biochemical methods to identify and confirm protein-protein interactions. IP uses a target protein-specific antibody conjugated with protein A/G affinity beads to identify molecules interacting with the target protein. Here, we describe the principle, procedure and challenges of the IP assay.
Since its discovery over three decades ago, signal transducer and activator of transcription 1 (STAT1) has been extensively studied as a central mediator for interferons (IFNs) signaling and ...antiviral defense. Here, using genetic and biochemical assays, we unveil Thr
as a conserved IFN-independent phosphorylation switch in Stat1, which restricts IFN signaling and promotes innate inflammatory responses following the recognition of the bacterial-derived toxin lipopolysaccharide (LPS). Genetically engineered mice expressing phospho-deficient threonine748-to-alanine (T748A) mutant Stat1 are resistant to LPS-induced lethality. Of note, T748A mice exhibited undisturbed IFN signaling, as well as total expression of Stat1. Further, the T748A point mutation of Stat1 recapitulates the safeguard effect of the genetic ablation of
following LPS-induced lethality, indicating that the Thr
phosphorylation contributes inflammatory functionalities of Stat1. Mechanistically, LPS-induced Toll-like receptor 4 endocytosis activates a cell-intrinsic IκB kinase-mediated Thr
phosphorylation of Stat1, which promotes macrophage inflammatory response while restricting the IFN and anti-inflammatory responses. Depletion of macrophages restores the sensitivity of the T748A mice to LPS-induced lethality. Together, our study indicates a phosphorylation-dependent modular functionality of Stat1 in innate immune responses: IFN phospho-tyrosine dependent and inflammatory phospho-threonine dependent. Better understanding of the Thr
phosphorylation of Stat1 may uncover advanced pharmacologically targetable molecules and offer better treatment modalities for sepsis, a disease that claims millions of lives annually.
Nonalcoholic fatty liver disease (NAFLD) is a complicated disease linked with dietary habitats, obesity, and a range of comorbidities correlated with insulin resistance.
Although environmental ...parameters are essential in deciding risk of the disease, proofs from previous reports sustain the hypothesis that genetics are responsible for NAFLD developmentand progression. Plasma cell antigen-1 (PC-1) and its gene polymorphism are associated with NAFLD progression. Consequently, the object of this study was to detect the usefulness of PC-1 K121Q gene polymorphism in NAFLD progression.
A total of 87 NAFLD patients were included in the study and subdivided ultrasonographically into 31 patients with grade 1 (mild NAFLD), 26 patients with grade 2 (moderate NAFLD) and 30 patients with grade 3 (severe NAFLD), in addition to 47 normal controls. The detection of PC-1 K121Q gene polymorphism was accomplished by using restriction fragment length polymorphism (RFLP)-PCR.
Lipid profile parameters were associated with the incidence of NAFLD. AlthoughPC-1 gene polymorphism didnot significantly change in parallel with NAFLD grades, PC-1 at the genetic and protein level was significantly associated with triacylglycerollevels in NAFLD patients.
Lipid profile indices are risk factors for the incidence of NAFLD. Triacylglycerol (TAG) level is the hall-mark in the NAFLD pathogenesis and in the predisposition of PC-1 gene polymorphism.
Nonalcoholic fatty liver disease (NAFLD) is a silent disease; its spectrum includes simple steatosis, nonalcoholic steatohepatitis and fibrosis. Pro- and anti-inflammatory cytokines play roles in the ...pathogenesis of NAFLD and insulin resistance (IR). Moreover, plasma cell antigen-1 (PC-1) is related to IR and associated with NAFLD progression. Therefore, we aimed to detect biomarkers, ultrasonographic and anthropometric findings capable of differentiating NAFLD grades, since most previous investigators were concerned more with NAFLD patients without classifying them into grades.
A total of 87 NAFLD patients (31 with grade 1 (mild NAFLD), 26 with grade 2 (moderate NAFLD) and 30 with grade 3 (severe NAFLD) were included in the study, in addition to 47 controls (grade 0). All subjects underwent ultrasonographic examination for NAFLD diagnosis. Serum interleukin-10 (IL-10), plasma interleukin-18 (IL-18) and plasma PC-1 levels were determined using enzyme-linked immunosorbent assay.
Homoeostasis model assessment (HOMA)-IR was higher in different NAFLD grades than in controls. Ultrasonographic and anthropometric findings and lipid profile indices (except for high-density lipoprotein cholesterol, which was decreased) were increased with NAFLD progression. Grade 3 patients showed significant increase in levels of IL-18 and significant decrease in IL-10 and PC-1 levels when compared to grade 1 patients.
Anthropometric and ultrasonographic findings were valuable in differentiating NAFLD grades. IR is very important in NAFLD pathogenesis. IL-18, HOMA-index and PC-1 levels could be used to differentiate between NAFLD grades, together with other measurements.
PDF
