SLC8 family of sodium/calcium exchangers in GtoPdb v.2021.3

The sodium/calcium exchangers (NCX) use the extracellular sodium concentration to facilitate the extrusion of calcium out of the cell. Alongside the plasma membrane Ca2+-ATPase (PMCA) and sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA), as well as the sodium/potassium/calcium exchangers (NKCX, SLC24 family), NCX allow recovery of intracellular calcium back to basal levels after cellular stimulation. When intracellular sodium ion levels rise, for example, following depolarisation, these transporters can operate in the reverse direction to allow calcium influx and sodium efflux, as an electrogenic mechanism. Structural modelling suggests the presence of 9 TM segments, with a large intracellular loop between the fifth and sixth TM segments [1].

Inside the Insulin Secretory Granule

The pancreatic β-cell is purpose-built for the production and secretion of insulin, the only hormone that can remove glucose from the bloodstream. Insulin is kept inside miniature membrane-bound storage compartments known as secretory granules (SGs), and these specialized organelles can readily fuse with the plasma membrane upon cellular stimulation to release insulin. Insulin is synthesized in the endoplasmic reticulum (ER) as a biologically inactive precursor, proinsulin, along with several other proteins that will also become members of the insulin SG. Their coordinated synthesis enables synchronized transit through the ER and Golgi apparatus for congregation at the trans-Golgi network, the initiating site of SG biogenesis. Here, proinsulin and its constituents enter the SG where conditions are optimized for proinsulin processing into insulin and subsequent insulin storage. A healthy β-cell is continually generating SGs to supply insulin in vast excess to what is secreted. Conversely, in type 2 diabetes (T2D), the inability of failing β-cells to secrete may be due to the limited biosynthesis of new insulin. Factors that drive the formation and maturation of SGs and thus the production of insulin are therefore critical for systemic glucose control. Here, we detail the formative hours of the insulin SG from the luminal perspective. We do this by mapping the journey of individual members of the SG as they contribute to its genesis.

Differential levels of IFNα subtypes in autoimmunity and viral infection

Type I interferons are essential for host response to viral infections, while dysregulation of their response can result in autoinflammation or autoimmunity. Among IFNα (alpha) responses, 13 subtypes exist that signal through the same receptor, but have been reported to have different effector functions. However, the lack of available tools for discriminating these closely related subtypes, in particular at the protein level, has restricted the study of their differential roles in disease. We developed a digital ELISA with specificity and high sensitivity for the IFNα2 subtype. Application of this assay, in parallel with our previously described pan-IFNα assay, allowed us to study different IFNα protein responses following cellular stimulation and in diverse patient cohorts. We observed different ratios of IFNα protein responses between viral infection and autoimmune patients. This analysis also revealed a small percentage of autoimmune patients with high IFNα2 protein measurements but low pan-IFNα measurements. Correlation with an ISG score and functional activity showed that in this small sub group of patients, IFNα2 protein measurements did not reflect its biological activity. This unusual phenotype was partly explained by the presence of anti-IFNα auto-antibodies in a subset of autoimmune patients. This study reports ultrasensitive assays for the study of IFNα proteins in patient samples and highlights the insights that can be obtained from the use of multiple phenotypic readouts in translational and clinical studies.

SLC8 family of sodium/calcium exchangers (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

The sodium/calcium exchangers (NCX) use the extracellular sodium concentration to facilitate the extrusion of calcium out of the cell. Alongside the plasma membrane Ca2+-ATPase (PMCA) and sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA), as well as the sodium/potassium/calcium exchangers (NKCX, SLC24 family), NCX allow recovery of intracellular calcium back to basal levels after cellular stimulation. When intracellular sodium ion levels rise, for example, following depolarisation, these transporters can operate in the reverse direction to allow calcium influx and sodium efflux, as an electrogenic mechanism. Structural modelling suggests the presence of 9 TM segments, with a large intracellular loop between the fifth and sixth TM segments.

Inducible expression of immediate early genes is regulated through dynamic chromatin association by NF45/ILF2 and NF90/ILF3

ABSTRACTImmediate early gene (IEG) transcription is rapidly activated by diverse stimuli without requiring new protein synthesis. This transcriptional regulation is assumed to involve constitutively expressed nuclear factors that are targets of signaling cascades initiated at the cell membrane. NF45 and its heterodimeric partner NF90 are chromatin-interacting proteins that are constitutively expressed and localized predominantly in the nucleus. Previously, NF90 chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) in K562 erythroleukemia cells revealed its enriched association with chromatin at active promoters and strong enhancers. NF90 specifically occupied the promoters of IEGs. Here, ChIP in serum-starved HEK293 cells demonstrated that NF45 and NF90 pre-exist and specifically occupy the promoters of IEG transcription factors EGR1, FOS and JUN. Cellular stimulation with phorbol myristyl acetate increased NF90 occupancy, while decreasing NF45 occupancy at promoters of EGR1, FOS and JUN. In HEK293 cells stably transfected with doxycycline-inducible shRNA vectors targeting NF90 or NF45, doxycycline-mediated knockdown of NF90 or NF45 attenuated the inducible expression of EGR1, FOS, and JUN at the levels of mRNA and protein. NF90 and NF45 operate as constitutively-expressed transcriptional regulators of IEGs. Dynamic chromatin association of NF45 and NF90 at IEG promoters are observed upon stimulation, and NF45 and NF90 contribute to inducible expression of IEGs. NF45 and NF90 operate as chromatin regulators of the immediate early response.

Image-Based Profiling Can Discriminate the Effects of Inhibitors on Signaling Pathways under Differential Ligand Stimulation

A major advantage of image-based phenotypic profiling of compounds is that numerous image features can be sampled and quantitatively evaluated in an unbiased way. However, since this assay is a discovery-oriented screening, it is difficult to determine the optimal experimental setup in advance. In this study, we examined whether variable cellular stimulation affects the efficacy of the image-based profiling of compounds. Seven different epidermal growth factor receptor (EGFR) ligands were used, and the expression of EGFR signaling molecules was monitored at various time points. Significant quantitative differences in image features were detected among the differentially treated samples. Next, 14 different compounds that affect EGFR signaling were profiled. Nearly half of the compounds were classified into distinct clusters, irrespective of differential ligand stimulation. The results suggest that image-based phenotypic profiling is quite robust in its ability to predict compound interaction with its target. Although this method will have to be validated in other experimental systems, the robustness of image-based compound profiling demonstrated in this work provides a valid basis for further study and its extended application.

Image-based profiling can discriminate the effects of inhibitors on signaling pathways under differential ligand stimulation

A major advantage of image-based phenotypic profiling of compounds is that numerous image features can be sampled and quantitatively evaluated in an unbiased way. However, since this assay is a discovery-oriented screening, it is difficult to determine the optimal experimental set-up in advance. In this study, we examined whether variable cellular stimulation affects the efficacy of image-based profiling of compounds. Seven different EGF receptor ligands were used, and the expression of EGF receptor signaling molecules was monitored at various time points. Significant quantitative differences in image features were detected among the differentially treated samples. Next, 14 different compounds that affect EGF receptor signaling were profiled. Nearly half of the compounds were classified into distinct clusters, irrespective of differential ligand stimulation. The results suggest that image-based phenotypic profiling is quite robust in its ability to predict compound interaction with its target. Although this method will have to be validated in other experimental systems, the robustness of image-based compound profiling demonstrated in this work provides a valid basis for further study and its extended application.