The Role of NLRP1, NLRP3, and AIM2 Inflammasomes in Psoriasis: Review

Inflammasomes are high-molecular-weight protein complexes that may cleave the two main proinflammatory cytokines, pro-interleukin-1β and pro-interleukin-18, into active forms, and contribute to psoriasis. Despite recent advances made in the pathogenesis of psoriasis, mainly studied as an autoimmune condition, activation of immune response triggers of psoriasis is still not completely understood. Recently, focus was placed on the role of inflammasomes in the pathogenesis of psoriasis. Multiple types of inhibitors and activators of various inflammasomes, inflammasome-related genes, and genetic susceptibility loci were recognized in psoriasis. In this systemic review, we collect recent and comprehensive evidence from the inflammasomes, NLRP1, NLRP3, and AIM2, in pathogenesis of psoriasis.

Obtaining peptides from aquatic organisms of the Antarctic region

The rapid growth in demand for peptide drugs is actualizing the search for new natural and economically viable sources of raw materials. The wide variety of biologically active compounds, including peptide nature, inherent in marine aquatic organisms allows us to consider their promising raw material resource. However, the use of marine objects as a source for the production of target molecules requires some optimization of existing methodological approaches to their isolation and ensure the appropriate degree of purification. The problem of optimization of the method of obtaining peptides from hydrobionts of the Antarctic region on the example of the hydrobiont Nacellaconcinna is solved in the work. The proposed three-step approach allows to isolate fractions of peptides of different molecular weight. The first step involved the precipitation of high molecular weight protein material first with perchloric acid and then with 80% ethyl alcohol. The result was a fraction of peptide molecules with a molecular weight of up to 6.5 kDa, which contained a small amount of high molecular weight protein impurities. Further purification of the obtained fraction was performed by ultrafiltration using membranes with a pore size of 10 kDa. Control of the protein-peptide composition of the sample at all stages of production was performed by disk electrophoresis under denaturing conditions in plates of 18% polyacrylamide gel. Analysis using 2D electrophoresis found that the isoelectric points of most peptides are in the pH range from 8.0 to 10.0. Only a small proportion of the peptides had isoelectric points at 4.0 and 5.0 pH. The final step of obtaining the peptide fraction involved fractionation of the sample by gel chromatography. As a result of chromatographic separation, four peaks were obtained, corresponding to the fractions with peptides, the molecular weight of which is about 2.3 kDa (1 peak), 1.9 kDa (2 peak), 1.4 kDa (3 peak) and 0.7 kDa (4 peak).

Optimization of western blotting for the detection of proteins of different molecular weight

Protein samples electroblotted onto nitrocellulose membranes and quenched with a mixture of blocking agents produced a strong signal for cystic fibrosis transmembrane-conductance regulator (CFTR), a high-molecular-weight protein, in western blotting. Optimized conditions for CFTR were then extended to medium- and low-molecular-weight proteins (LAMP1 and Rab11a, respectively) to determine the effects of methanol concentration (0–20%) in Towbin’s transfer buffer (TTB). Methanol in TTB appears to have little to no effect on CFTR signal. However, for medium-sized (LAMP1) and small (Rab11a) proteins, a lower concentration of methanol (10%) was sufficient to produce a maximal signal. Therefore, methanol, a toxic solvent, can be removed from or reduced in TTB without compromising signal strength. Here, we show modifications that may be useful in detecting and/or improving the signal of low-abundance proteins.

Biochemical and molecular characterisation of the proposed P2Y1-P2Y12 receptor heterodimer in tsA201 cells

The P2Y receptor family represents a wide-ranging set of G-protein-coupled receptors that respond to purinergic ligands, and are involved in many physiological processes. From signalling assays in tsA201 cells, the Kennedy lab has previously proposed the formation of a constitutive heterodimer between co-expressed human P2Y1 and P2Y12 receptors; this could represent a target for the control of pain sensing neurones. Therefore, it was aimed in this project to characterise if the receptors physically interacted using co-immunoprecipitation, and also to investigate the endogenous expression of other potentially interacting hP2Y receptors in the tsA201 cell line. In this study, tsA201 cells were determined to express mRNA for hP2Y1, 2, 4, 6, 12, 14 receptors using reverse transcriptase PCR and confirmatory Sanger sequencing. Furthermore, the cells demonstrated Ca2+ responses to all of the natural hP2Y receptor agonists tested, the order of potency being ADP > ATP > UTP > UDP. When co-expressing hP2Y1 and hP2Y12 receptors oppositely tagged with a HA or fluorescent protein (FP), immunoprecipitating for the HA tag and blotting for the FP showed a visible high molecular weight protein complex of ~130 to >250 KDa for either combination of receptors. This implied that the FP tagged receptor had co-immunoprecipitated with the other HA tagged receptor as this protein was also visible when blotting for the HA tag, signifying the presence of both tagged receptors in the complex. These results were overall suggestive of functional hP2Y1, 2, 12 receptor expression in the tsA201 cell line, and possibly also hP2Y4, 6, 14 receptors. The high molecular weight protein complex detected could represent a constitutive hP2Y1-hP2Y12 receptor heterodimer as proposed. Whilst it was not possible to specifically either confirm or refute the physical formation of the heterodimer with the data acquired, it was potentially supportive, and furthermore suggested other future paths of investigation.